Laboratory Notebook

Note before you start reading

Instead of using the iGEM parts’ nomenclature (BBa_…) to name the sequences, in the following Laboratory Notebook section, we are using the following abbreviations:

sequence 1: BBa_K228005
sequence 2: BBa_K228006
sequence 3: BBa_K228007
sequence 4: BBa_K228008
sequence 5: BBa_K228009
sequence 6: BBa_K228010
sequence 7: BBa_K228011
sequence 8: BBa_K228012
sequence 9: BBa_K228013

Introduction

Here you will find the complete report of all our experiments since the 24/08. We decided to not post the first two months of our experiments because they were mostly optimisation protocols. We reached the lab at the end of June without any experience in synthetic biology and few in molecular biology, acquired from diverse internships and courses. This world was new to us so a lot of troubles awaited us. We had many troubles with PCR and ligations because of diverse reasons that we understood as the days were going on.

However, a lot of positive things are to be retained from this preliminary work. We learnt how to organise ourselves properly, how to optimise protocols according to our lab (and with the precious help of our advisors), and we learnt also to persevere.

Most of all, science is built on failure, defined and molded by it. What is most important thing for us is that although we had many difficulties, we continued no matter what. In the end we managed to make our sequence work, and that was very rewarding. We are aware some of you could learn from the experience we got at La Paillasse, therefore don’t hesitate to contact us by mail so we can share our acquired knowledge.

You can now consult our experiments, the 08/24 began with a digestion of the BBA_J04450 part in order to use its backbone pSB1C3 for the ligation of our sequences acquired by PCR.

Table of content

Week 1

08/24 - Digestion of pSB1C3 - mRFP

Objective:

To digest the plasmid pSB1C3-mRFP with the two restriction enzymes EcoRI and PstI in order to separate the backbone and the insert.

Material:

Plasmid pSB1C3-mRFP (psB1C3-BBa_J04450 from the IGEM Distribution Kit)

EcoRI-HF (NEB: 20000 U/mL)

PstI (NEB: 20000 U/mL)

10X NEB buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

The pSB1C3-mRFP plasmid was digested according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
PstI	1µL	40U/µL
pSB1C3-mRFP	10µL	10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	33µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

For the migration (cf 08/24 - PCR of sequence 2 with the TATA primers), we used 2/4/8µl of the digestion product to quantify the Backbone concentration efficiently.

08/24 - PCR of sequence 2 (BBa_K2282006) with the TATA primers

Objective:

We received our TATA primers from IDT. Primers TATA Suffix and TATA prefix were first resuspended in 250µl of TE buffer each.

Both tubes were vortexed, heated at 50°C for 20 minutes and vortexed again.

A white precipitate was still observable. According to IDT, it could be silica or other by-products. Still we need to be careful.

Part 1: PCR

Material:

Forward primer (100µM) synthesized by IDT

Reverse primer (100µM) synthesized by IDT

DNA template (BBa_K2282006, synthesized by IDT)

Q5® High-Fidelity 2X Master Mix (NEB)

DNase/RNase free water

PCR tubes

Micropipettes + tips

Thermocycler

Protocol:

Pre-dilutions préparation:

Primers 100µM: 1 µL primer + 9 dH2O --> 10µM final

Tubes were prepared according to the following indications:

Tube	Annealing T°C	Primer Prefix (10µM)	Primer Suffix (10µM)	DNA template	Q5 Mix	dH2O DNase/RNase free	Total Vol. (µL)
C+	60°C	2,5µL	2,5µL	1µL pSB1C3-mRFP (miniprep)	12.5 µL	6,5µL	25
2	60°C	5µL	5µL	2 µL seq 2 (IDT)	25 µL	13µL	30

The tubes were put in the thermocycler with the following program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	98°C
30	Denaturation	10 sec	98°C
	Annealing	30 sec	60°C
	DNA polymerization	30 sec (25-30sec/kb)	72°C
0	DNA polymerization	2 min	72°C
	END	∞	4°C

For the positive control plasmid, DNA polymerisation was set at 72°C for 1min 30 sec because of its length (3 kb vs. 1 kb for seq2). PCR tubes were placed in different thermocyclers. → This was a mistake. We should have let 30sec for the positive control also, as the amplified fragment (mRFP) is of 1kb.

Part 2: Gel migration of seq 2 (BBa_K2282006) PCR + pSB1C3-mRFP digested

Material:

Agarose

Distilled water

Gel cast and combs

Electrophoresis cuve

Microwave

Micropipettes and tips

2-Log DNA Ladder (0.1-10.0 kb) (NEB)

Gel loading dye purple 6X (NEB)

Protocol:

Mixes preparation:

Sample	Product (µL)	6X Loading Dye (µL)	Total (µL)
seq 2 (from PCR)	7	1.4	8.4
C+ (positive control) (from PCR)	15	3	18
Digested pSB1C3-mRFP	15	3	18

Plan of deposit:

Ladder (5µL)
C+ - 15µL DNA (18µL of the mix)
seq 2 - 2µL DNA (2,4µL of the mix)
seq 2 - 4µL DNA (4,8µL of the mix)
pSB1C3-mRFP - 2µL (2,4µL of the mix)
pSB1C3-mRFP - 4µL (4,8µL of the mix)
pSB1C3-mRFP - 8µL (9,6µL of the mix)

Migration was set at 110V for 1 hour.

The gel was put inside a Gel Red bath for 1h30 and revealed under UV light (new bath: 50µL GelRed into 166mL dH2O).

Results:

Interpretation:

Bands were visible and their respective thickness were as expected. Except for the positive control, which did not work (weak smear).

Concentration estimations:

Seq 2 (BBa_K2282006): 50ng in 4µL

→ 475ng in 38µL

PCR cleanup* (-25%) → 356,25ng in 30µL

→ 11,875 ng/µL

pSB1C3 backbone: 70ng in 8µL

→ 306ng in 35µL

→ 8,75 ng/µL

*PCR cleanup was performed using the Qiagen kit from 2016.

Twice 15µL of DNase-free water were used for the final elution (30µL total).

08/24 - Digestion of seq 2 (BBa_K2282006) + pSB1C3 control and Ligation

Objective:

To digest the sequence 2 for subsequent ligation into pSB1C3-mRFP backbone, and make a ligation control by digesting pSB1C3-mRFP with EcoR1-HF endonuclease.

Part 1: Digestion

Material:

Plasmid pSB1C3-mRFP (BBa_J04450 from IGEM Kit)

PstI (NEB: 20000U/mL)

10X NEB Buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

We added the components of the mixes in the following order:

Reagents	Quantity
EcoRI-HF	1µL
pSB1C3-mRFP (from miniprep)	10µL
10X NEB buffer 2.1	5µL
Ultrapure water	34µL
Total volume	50µL

Reagents	Quantity
Purified seq 2 (11,875 ng/µL)	30µL
EcoRI-HF	1µL
PstI	1µL
10X NEB buffer 2.1	5µL (1X)
Ultrapure water	13µL
Total volume	50µL

Final seq 2 (BBa_K2282006) concentration: 7,125 ng/µL

We incubated for 2 hours at 37°C

Heat inactivation at 80°C for 20min

Part 2: Ligation

Material:

Digested insert (BBa_K2282006)

Digested backbone (pSB1C3 from IGEM kit)

10X T4 DNA Ligase Buffer (NEB)

T4 DNA ligase (400000U/mL)

Ultrapure water

Micropipettes and tips

Eppendorf tubes

Protocol:

We added the components of the mix in the following order:

Sample	01:01	01:03	01:05	1:3 *2	Ligase control 1	Ligase control 2
insert (7,125 ng/µL)	1,6µL	4,9µL	8,2µL	9,8µL	-	-
backbone pSB1C3 (8,75 ng/µL)	2,9µL	2,9µL	2,9µL	5,8µL	-	-
backbone control	-	-	-	-	5µL	5µL
10X T4 DNA Ligase Buffer	2µL	2µL	2µL	2µL	2µL	2µL
T4 DNA Ligase	2 µL	2 µL	2 µL	2 µL	2 µL	-
dH2O	11,5µL	8,2µL	4,9µL	0,4µL	11µL	13µL
Total volume	20µL	20µL	20µL	20µL	20µL	20µL

Incubation overnight at Room Temperature.

08/25 - Transformation seq 2 (BBa_K2282006)

Objective: To transform competent cells with the sequence 2 ligated into pSB1C3 backbone in order to amplify the recombinant plasmid.

Material:

Ligated plasmid (pSB1C3 + BBa_K2282006)

Competent cells (DH5-alpha)

LB medium liquid and solid

SOC medium

Chloramphenicol

Ice

Eppendorf tubes

Waterbath

Micropipettes and tips

Petri diches

Glass beads or inoculating loop

Incubator

Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Transformation	Plate	Competent cells
(1) 1:1	20µL	Yes	LB + Chloramphenicol	50µL
(2) 1:3	20µL	Yes	LB + Chloramphenicol	50µL
(3) 1:5	20µL	Yes	LB + Chloramphenicol	50µL
(4) 1:3 * 2	20µL	Yes	LB + Chloramphenicol	50µL
(5) Control Ligase	20µL	Yes	LB + Chloramphenicol	50µL
(6) Control -	20µL	Yes	LB + Chloramphenicol	50µL
(7) Control Transformation (miniprep)	10µL	Yes	LB + Chloramphenicol	50µL
(8) LB medium	-	No	LB	50µL

Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500 rpm for 4minutes and threw away most of the culture media. Bacteria were resuspended into the remaining culture media and the total volume was spread in the corresponding petri dishes.

Incubate at 37°C overnight or approximately 16 hours (we let 2 days). Position the plates with the agar side at the top, and the lid at the bottom.

Results:

Interpretation:

Results were as expected for the controls as for the ligation tests.

The different backbone/insert ratios for the ligation suggest that a ratio of 1:3 or 1:5 works better than 1:1 or 1:3 with double quantities.

Some backbones have ligated themselves spontaneously without ligase, but fewer colonies are observable for this condition showing the efficiency of ligase.

The presence of red colonies in the test conditions was predictable as we did not purify the backbone digestion products before performing the ligation. They correspond to the non-digested pSB1C3-mRFP backbones transformed into the bacteria, giving a red coloration. We will have to pick the isolated blue colonies in order to purify the pSB1C3-seq2 plasmid through culture and miniprep.

Week 2

08/28 - PCR on sequence 7 (BBa_K2282011); digestion pSB1A3-mRFP; PCR clean up; Digestion sequence 7; Ligations OverNight

Objective: To amplify sequence 7 in order to ligate it in pSB1A3 backbone, and then to transform DH5α.

Part 1: PCR

Material:

Forward primer (100µM) synthesized by IDT

Reverse primer (100µM) synthesized by IDT

DNA template (sequence 7 BBa_K2282011, synthesized by IDT)

Q5® High-Fidelity 2X Master Mix (NEB)

DNase/RNase free water

PCR tubes

Micropipettes and tips

Thermocycler

Protocol:

Pre-dilutions preparation:

Primers 100µM: 1 µL primer + 9 dH2O → 10µM final

Tubes	Primer Prefix (10µM)	Primer Suffix (10µM)	DNA template	Q5 Mix	dH2O DNase/RNase free	Total Volume (µL)
7	5µL	5µL	2 µL seq 7	25 µL	13 µL	50

The tubes were put in the thermocycler with the following program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	98°C
30	Denaturation	10 sec	98°C
	Annealing	30 sec	60°C
	DNA polymerization	30 sec (25-30sec/kb)	72°C
0	DNA polymerization	2 min	72°C
	END	∞	4°C

Part 2: Digestion of pSB1A3-mRFP

Objective: The goal here is to simply digest our plasmid PSB1A3-mRFP.

Material:

Plasmid pSB1A3-mRFP (from IGEM Kit)

EcoRI-HF (NEB: 20000U/mL)

PstI (NEB: 20000U/mL)

10X NEB Buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

We prepared the mix according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
PstI	1 µL	40U/µL
pSB1A3-mRFP	10 µL	10ng/µL
10X NEB buffer 2.1	5 µl	1X
Ultrapure water	33 µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

For the migration, we are going to use 2/4/8µL of the digestion product to quantify the Backbone concentration efficiently.

Part 3: Gel migration

Objective: To perform the migration of seq 7 fragment obtained from PCR and the digested pSB1A3-mRFP.

Material:

Agarose

Distilled water

TAE buffer 50X

Gel cast and combs

Electrophoresis cuve

Microwave

Micropipettes and tips

2-Log DNA Ladder (0.1-10.0 kb) (NEB)

Gel loading dye purple 6X (NEB)

Protocol:

We made an agarose gel. (cf our Protocol section)

We want to migrate the seq 7 (BBa_K2282011) from PCR and the pSB1A3-mRFP digested.

Mixes preparation:

Sample	Product (µL)	6X Loading dye (µL)	Total (µL)
seq 7 (from PCR)	7	1.4	8.4
Digested pSB1A3-mRFP	15	3	18

Plan of deposit:

1: Ladder (5µL)

2: seq 7 - 2µL DNA (2,4µL of the mix)

3: seq 7 - 4µL DNA (4,8µL of the mix)

4: pSB1A3-mRFP - 2µL (2,4µL of the mix) (this well has not been correctly filled up)

5: pSB1A3-mRFP - 4µL (4,8µL of the mix)

6: pSB1A3-mRFP - 8µL (9,6µL of the mix)

7: Ladder (5µL)

Migration 1h30 at 110V.

Revelation in GelRed bath during 1h30.

Results:

Interpretation:

Estimation of quantities:

Seq 7: 50ng in 4µL

→ 537.5ng in 43µL

PCR cleanup* (-25%) → 403ng in 50µL

→ 8.06 ng/µL

pSB1A3 backbone: 70ng in 8µL

→ 306ng in 35µL

→ 8,75 ng/µL

*cf Qiagen Kit protocol

Part 4: Digestion of sequence 7 (BBa_K2282011)

Objective: To digest the sequence 7 for subsequent ligation into pSB1A3-mRFP backbone, and make a ligation control by digesting pSB1A3-mRFP with PstI endonuclease.

Material:

Sequence 7 (BBa_K2282011, synthesized by IDT)

EcoRI-HF (NEB: 20000U/mL)

PstI (NEB: 20000U/mL)

10X NEB Buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

We prepare the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	80U/µL
PstI	1µL	80U/µL
purified sequence 7	30µL	8.06 ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	13µL

Reagents	Quantity	Final concentration
PstI	1µL	40U/µL
pSB1A3-mRFP	10µL	10 ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	34µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

Part 5: Ligation

Material:

Digested insert (seq 7 BBa_K2282011)

Digested backbone (pSB1A3)

10X T4 DNA Ligase Buffer (NEB)

T4 DNA ligase (400000U/mL)

Ultrapure water

Eppendorf tubes

Protocol:

We prepared the mixes according to the following instructions:

insert (4,836 ng/µL)	8µL	13.3µL	-	-
backbone pSB1A3 (8,75 ng/µL)	2.9µL	2.9µL	-	-
backbone control digested	-	-	5µL	5µL
10X T4 DNA Ligase Buffer	2µL	2µL	2µL	2µL
T4 DNA Ligase	1µl	1µL	1µL	-
Ultrapure water	6.1µL	0.8µL	12µL	13µL
Total volume	20µL	20µL	20µL	20µL

Ligation overnight in a room at about 16°C.

08/29 - Transformation with sequence 7

Objective: To transform competent cells with the sequence 7 ligated into pSB1A3 backbone in order to amplify the recombinant plasmid.

Material:

Ligated plasmid

Competent cells (DH5-alpha)

LB medium liquid and solid

SOC medium

Ampicillin

Ice

Eppendorf tubes

Micropipettes and tips

Waterbath

Petri dishes

Glass beads

Incubator

Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Transformation	Plate	Competent cells
1:3	20µL	Yes	LB + Ampicillin	50µL
1:5	20µL	Yes	LB + Ampicillin	50µL
Control ligase +	20µL	Yes	LB + Ampicillin	50µL
Control ligase -	20µL	Yes	LB + Ampicillin	50µL
Control Transformation (miniprep pSB1A3)	10µL	Yes	LB + Ampicillin	50µL
LB medium	-	No	LB	50µL

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. (IN THE FRIDGE AS WELL)

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and threw away most of the culture media.

Bacteria were resuspended into the remaining culture media and the total volume was spread in the corresponding petri dishes.

Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

Results:

Interpretation:

Controls worked. However there is not a lot of colonies in the dish. Thus we can imagine that there is no lot of bacteria transformed with the seq 7.

08/30 - Transformation of AmilCP (BBa_K592009) from iGEM kit and sequence 2 (BBa_K2282006)

Objective: To transform competent cells with the iGEM plasmid pSB1C3-AmilCP (BBa_K592009) and sequence 2 (BBa_K2282006) ligated into pSB1C3 on the 24/08 (miniprep performed on 30/08) in order to compare the expression of AmilCP at high and low temperatures.

Note: The constitutive promoters and the RBS we used were different between the two plasmids. Therefore, comparing their respective protein quantity or kinetics of appearance would be irrelevant. This assay aims at ensuring that the presence of the DS box into the AmilCP sequence does not alter its coloration.

Material:

Ligated plasmid (sequence 2-pSB1C3)

IGEM plasmid pSB1C3-amilCP (BBa_K592009)

Competent cells (DH5-alpha)

LB medium liquid and solid

SOC medium

Ampicillin

Ice

Eppendorf tubes

Micropipettes and tips

Waterbath

Petri dishes

Glass beads

Incubator

Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Transformation	Plate	Competent cells	SOC medium
pSB1C3-amilCP (from iGEM kit)*	3µL	Yes	LB + Chloramphenicol	50µL	947µL
Seq 2 (from the 30/08 miniprep)	10µL	Yes	LB + Chloramphenicol	50µL	940µL

*The plasmid was taken from the DNA distribution kit plate given by the iGEM (plate 6 well 1N). The DNA was resuspended into 10µL of dH20 to reach a final concentration of approximately 200-300 pg/µL.

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and 850µL of supernatant was discarded. Bacteria were resuspended into the remaining culture media and bacteria were spread in the corresponding petri dishes as follows:

Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

Results:

Results (pictures were taken after 5 days):

Interpretation:

Blue colonies were observable with the recombinant pSB1C3-seq2 plasmid (containing amilCP + DSbox). This shows that the miniprep performed the same day has worked properly, and that this isolated plasmid can be used in further experiments.

Colonies transformed with the plasmid from iGEM distribution kit plates (containing amilCP without DS box) did not grow very well: only one colony was observable in one of the two dishes. This can be explained by the fact that only 600pg (in average) of DNA was used for the transformation, whereas the first plasmid came from a miniprep that is known to give a better yield.

However, one colony was picked up from each condition on the friday and was cultured in liquid medium over the week-end. This will serve as a comparison experiment to assess if any difference is observable in the blue coloration (absorbance at 588nm). Given the fact that the two plasmids do not carry the same promoter and RBS sites, and therefore induce a slightly differential expression level of the protein, we can only use the iGEM plasmid to compare the color characteristics.

08/31 - PCR colony on sequence 7 (BBa_K2282011)

Objective: To check if our sequence 7 (BBa_K2282011) is present in our colonies.

Material:

10X Standard taq reaction Buffer (NEB)

10µM dNTPS (NEB)

Forward primer 100µM (synthesized by IDT)

Reverse primer 100µM (synthesized by IDT)

Taq DNA polymerase (5000U/mL)

DNAse/ RNAse free water

PCR tubes

Thermocycler

Micropipettes and tips

Protocol:

Part 1: PCR colony

Mix preparation:

We have 11 tubes of 25µL to test. We prepared a pre-mix for a final volume of 300µL.

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	30 µL	1X
10mM dNTPs	6 µL	200µM (1/50)
100µM Prefix Primer	3 µL	1µM
100µM Suffix Primer	3 µL	1µM
Taq DNA Polymerase	3 µL	2,5 units/50µL PCR
Nuclease-free water	255 µL	qs 300 µL

Add the colonies from the plates:

1:5
1:5
1:5
1:5
1:3
1:3
1:3
1:3
pSB1A3-mRFP
white colony from ligation of seq 2, 1:3 (25/08)
non transformed bacteria (from antibiotic-free dish, 25/08)

At the same time we introduce a part of each colony (from 1:5 and 1:3) in 1mL liquid LB medium containing ampicillin (100µg for 1mL). We put the tubes in the incubator at 37°C, 200rpm shaking.

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.

Put the PCR tubes in the thermocycler and run the following program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	5 min	95°C
30	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (25-30sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	END	∞	4°C

Part 2: Gel migration

Agarose

Distilled water

Gel cast and combs

Electrophoresis cuve

Microwave

Micropipettes and tips

2-Log DNA Ladder (0.1-10.0 kb) (NEB)

Gel loading dye purple 6X (NEB)

Protocol:

Make an agarose gel (cf our "Protocol" section)

Mixes preparation: 1µL 6X Loading Dye + 5µL sample from PCR

Plan of deposit:

Ladder (5µL)
1:5_1 (6µL)
1:5_2 (6µL)
1:5_3 (6µL)
1:5_4 (6µL)
1:3_5 (6µL)
1:3_6 (6µL)
1:3_7 (6µL)
1:3_8 (6µL)
pSB1A3-mRFP (6µL)
white colony (6µL)
non transformed (6µL)
Ladder (5µL)

Migration during 1h30 at 110V.

GelRed bath overnight.

Results:

There is nothing on the gel.

Interpretation:

We make several suppositions in order to explain our results;

- The concentrations is maybe too low to be visible

- Sequence 7 has maybe not been amplified during the PCR (PCR failed)

- Ligation didn't work efficiently for sequence 7

- Transformation didn't work efficiently for sequence 7

- PCR colony didn't work

Also one of the Ladder is missing, maybe it is due to a mistake during the deposit.

HOWEVER we can distinguish a little light for the pSB1A3-mRFP. It could confirm that PCR colony worked.

09/01 - PCR sequence 7 (BBa_K2282011) & Protegrin-1 (BBa_K628000)

Objective: Amplify our cold-shock sequence 7 to further ligate it into pSB1C3.

Material:

Forward primer (100µM) synthesized by IDT

Reverse primer (100µM) synthesized by IDT

DNA template (sequence 7: BBa_K2282011 from IDT and protegrin-1: BBa_K628000 from iGEM kit

Q5® High-Fidelity 2X Master Mix (NEB)

10X Standard taq reaction Buffer (NEB)

10µM dNTPS (NEB)

Taq DNA polymerase (5000U/mL)

DNase/RNase free water

PCR tubes

Micropipettes and tips

Thermocycler

Agarose

Distilled water

TAE buffer 50X

Gel cast and combs

Electrophoresis cuve

Microwave

2-Log DNA Ladder (0.1-10.0 kb) (NEB)

Gel loading dye purple 6X (NEB)

Protocol:

Tube Prot-1

Pre-dilutions preparation:

Primers 100µM: 1 µL primer + 9 µL dH2O --> 10µM final

Tubes	Primer Prefix (10µM)	Primer Suffix (10µM)	DNA template	Q5 Mix	dH2O DNase/RNase free	Total Volume
Prot-1	5 µL	5 µL	2 µL	25 µL	13 µL	50 µL

Tube Sequence 7

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	1 µL	9 µL	500 U/mL
dNTPs	1 µL	9 µL	1 mM

We then prepare the mix according to the following instructions:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	2.5 µL	1X
1mM dNTPs	5 µL	200µM (1/50)
10µM Prefix Primer	2.5 µL	1µM
10µM Suffix Primer	2.5 µL	1µM
Taq DNA Polymerase 500U/mL	2.5 µL	1,25 units/25µL PCR
DNA template (10ng/µL)	1 µL
Nuclease-free water	10 µL	qs 25 µL

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.

The tubes were put in the thermocycler with the following program:

Tube Prot-1

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	98°C
30	Denaturation	10 sec	98°C
	Annealing	30 sec	60°C
	DNA polymerization	20 sec (25-30sec/kb)	72°C
0	DNA polymerization	2 min	72°C
	END	∞	4°C

Tube Sequence 7

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	5 min	95°C
30	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (25-30sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	END	∞	4°C

Migration

Sample preparation:

for PCR colony samples of 08/31: 20µl sample + 4µl 6X Loading dye

for PCR of 09/01: 5µl sample + 1µl 6X Loading dye

Deposit plan:

Ladder (5µL)
1:5_1 (24µL)
1:5_2 (24µL)
1:5_3 (24µL)
1:5_4 (24µL)
1:3_5 (24µL)
1:3_6 (24µL)
1:3_7 (24µL)
1:3_8 (24µL)
pSB1A3-mRFP (24µL)
white colony (24µL)
non transformed (24µL)
Prot-1 (24µL)
seq 7 (24µL)
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

GelRed bath during 1h30.

Results:

Interpretation:

Protegrin-1 is maybe out of the gel (because the sequence is too small, 60bp).

Seq 7 amplified by Taq did not work (while it seems to have been efficient on some colonies and pSB1A3-mRFP).

There is nothing for not transformed colonie and the white colonie from seq 2 dish, as expected.

Our problem is to differentiate mRFP and seq 7 which have a very similar length.

Week 3

09/04 - PCR on sequences 8 (BBa_K2282012) and 9 (BBa_K2282013)

Objective: To amplify our heat-shock sequences 8 and 9 in order to further ligate them into our recombinant pSB1C3-Seq2 plasmid (containing AmilCP and the DS box).

Material:

Forward primer (100µM) synthesized by IDT

Reverse primer (100µM) synthesized by IDT

DNA template (BBa_K2282012 and BBa_K2282013)

Q5® High-Fidelity 2X Master Mix (NEB)

DNase/RNase free water

PCR tubes

Micropipettes and tips

Thermocycler

Protocol:

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Pre-dilutions (1/10):

	Samples	dH2O DNase/RNase free	Final concentration
Taq Polymerase	1.5 µL	13.5 µL	500 U/mL
Primers (100µM)	1.5 µL	13.5 µL	10 µM

We prepared the mix for 25µl PCR mix volume (5 tubes: 2 x seq8, 2 x seq9, 1 x control pSB1C3-mRFP) according to the following instructions:

Components	Volume	Final concentration
10X Standard Taq Reaction Buffer	2.5 µL	1X
1mM dNTPs	0.5 µL	200µM (1/50)
10µM Prefix Primer	2.5 µL	1µM
10µM Suffix Primer	2.5 µL	1µM
Taq DNA Polymerase 500U/mL	2.5 µL	1,25 units/25µL PCR
DNA template (10ng/µL)	1 µL
Nuclease-free water	13.5 µL	qs 25 µL

The PCR mixes are divided in two tubes of 25µl for each sample.

The tubes were put in the thermocycler with the following program:

Seq 8 and control pSB1C3-mRFP:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (30sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	END	∞	4°C

Seq 9:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	2 min (30sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	END	∞	4°C

We decided to perform 18 cycles instead of 30 to reduce the risk of error.

09/04 - Digestion of pSB1C3-Seq2 (BBa_K2282006)

Objective: The goal here is to simply digest our plasmid pSB1C3-Seq 2 from a miniprep of the 08/28.

Material:

Plasmid pSB1C3-seq2 (BBa_K2282006)

EcoRI-HF (NEB: 20000U/mL)

PstI (NEB: 20000U/mL)

10X NEB Buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

We prepare the mix for the digestion of pSB1C3-Seq 2 according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
PstI	1 µL	40U/µL
pSB1C3-Seq 2	10 µL	~10ng/µL
10X NEB buffer 2.1	5 µl	1X
Ultrapure water	33 µL

We proceeded 2h at 37°C and heat inactivated for 20 min at 80°C.

09/05 - Gel migration of sequences 8 and 9 from 09/04 PCR; digestion; ligation

Part 1: Gel Migration

Objective: To run a gel with the PCR samples (seq 8, 9, ctrl) from the PCR performed with Taq polymerase on the previous day.

Material:

Agarose

Distilled water

TAE buffer 50X

Gel cast and combs

Electrophoresis cuve

Microwave

Micropipettes and tips

2-Log DNA Ladder (0.1-10.0 kb) (NEB)

Gel loading dye purple 6X (NEB)

Protocol:

Gel preparation: please see the Protocol section

Sample preparation: We put together the two tubes of sequence 8 in one single tube (Vf = 50µL). Same for sequence 9. For the sample preparation we ue 5µL of sample + 1µL of 6X Loading Dye.

Deposit plan:

Ladder (5µL)
Seq 8 (6µL)
Seq 9 (6µL)
Ctrl (pSB1C3-mRFP) (6µL)

Gel migration was performed during 1h30 at 110V.

GelRed bath during 1h30.

Results:

Interpretation:

Results are as we expected: Seq 8 at 1000 bp, Seq 9 at 2000 bp and Control at 1000 bp.

Part 2. PCR clean-up

Objective: To clean our PCR products.

Material:

QIAquick PCR purification kit (Quiagen)

PCR products

Eppendorf tubes

Deionised water

Micropipettes and tips

Centrifuge

Protocol: (see the Protocol section)

Elution was performed with deionised water: 15µL twice, for a total volume of 30µL.

Estimations of DNA concentration thanks to the gel:

Seq8: For 5µL → 30ng so for 45µL → 270ng so after PCR clean up (25% lost) → 202,5ng (6.75 ng/µL)

Seq9: For 5µL → 45ng so for 45µL → 405ng so after PCR clean up (25% lost) → 303,75ng (10.13 ng/µL)

Part 3. Digestion

Objective: to digest our sequences in order to perform the ligation.

Material:

Amplified and purified sequences (8 = BBa_K2282012 and 9 = BBa_K2282013)

EcoRI-HF (NEB: 20000U/mL)

PstI (NEB: 20000U/mL)

10X NEB Buffer 2.1

Ultrapure water

1,5mL Eppendorf tubes

Micropipettes + tips

Waterbath

Heat Block

Protocol:

We prepared the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
PstI	1 µL	40U/µL
sequence (8 or 9)	30 µL	6,7ng/µL (seq8) ; 10,1ng/µL (seq9)
10X NEB buffer 2.1	5 µl	1X
Ultrapure water	13 µL

We incubated 2h at 37°C.

Heat inactivation was performed at 80°C for 20min.

Part 4. Ligation

Objective: to ligate our sequences within the backbone.

Material:

Digested insert (Sequence 8 BBa_K2282012 and sequence 9 BBa_K2282013)

Digested backbone (pSB1C3-seq2)

10X T4 DNA Ligase Buffer (NEB)

T4 DNA ligase (400000U/mL)

Ultrapure water

Eppendorf tubes

Micropipettes and tips

Protocol:

Here are the estimated quantities of DNA we have (estimation from the gel):

Seq 8: 6,7ng/µL

Seq 9: 10,1ng/µL

Backbone: 16ng/µL

We prepare the ligation mixes according to the following instructions:

	seq 8 ratio 1:3	seq 8 ratio 1:5	seq 9 ratio 1:3	seq 9 ratio 1:5
insert	6,2µL	10.4µL	7.15µL	11.9µL
backbone pSB1C3-Seq 2 (16ng/µL)	1.5µL	1.5µL	1.5µL	1.5µL
10X T4 DNA Ligase Buffer	2µL	2µL	2µL	2µL
T4 DNA Ligase	1µL	1µL	1µL	1µL
dH2O	9.3µL	5.1µL	8.35µL	3.6µL
Total volume	20µL	20µL	20µL	20µL

Ligation overnight in a room at about 16°C.

09/06 - Bacterial transformation for sequences 8 and 9

Objective: To introduce our recombinant plasmids pSB1C3-seq 8 and pSB1C3-seq 9 in E. coli and amplify them.

Material:

Ligated plasmid (pSB1C3-seq 8 and pSB1C3-seq9)

Competent cells (DH5-alpha)

LB medium liquid and solid

SOC medium

Chloramphenicol

Ice

Eppendorf tubes

Micropipettes and tips

Waterbath

Petri dishes

Glass beads

Incubator

Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 45 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour and 20 min shaking at 200-300rpm (250rpm).

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and threw away most of the culture media. Bacteria were resuspended into the remaining culture media and the total volume was spread in the corresponding petri dishes.

Incubate at 30°C and 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

Results:

Pictures were taken 9 days after transformation.

.

Interpretation:

We can observe that we obtained red and white colonies. The red ones might have our sequences of interest (they may contain mRFP part).

09/08 - Liquid culture of transformed colonies

Objective: The goal here was to make liquid cultures of transformed colonies of the 06/09 to test out temperatures effects on sequences 8 and 9.

Material:

LB medium with chloramphenicol

Transformed colonies (on solid LB medium)

Sterile loops

Incubator with shaker (25°C and 37°C)

Sterile falcon 50mL

Protocol:

8 falcon tubes of 15ml of LB medium with chloramphenicol were prepared:

2 pSB1C3-mRFP at 25°C and 37°C

2 pSB1C3-seq 8 at 25°C and 37°C

2 pSB1C3-seq 9 at 25°C and 37°C

1 pSB1C3-seq 1 at 37 °C

1 pSB1C3-seq 2 at 37 °C

Both incubator were set at 250 rpm, however the multitron (used for 25°C condition) is a multiplate shaker.

Results:

Nothing grew in the pSB1C3-mRFP tubes, highlighting the fact that the colonies chosen were probably dead (the petri dish was outdated).

Both seq 8 and 9 displayed red color at 37°C, which is a logical result.

None of the seq 8 and 9 displayed colors at 25°C, however the shaking might have disturbed and considerably stressed the bacteria, hence nothing can really be interpreted from the results.

Both seq 1 and 2 colonies grew but no color was observed.

Protocol (continuation):

Aliquots of 1ml were made with every tube at 25°C and the seq 1 and seq 2 tubes at 37°C, and centrifuged at 10000 rpm for 1 minute.

Results:

Blue was observed for the seq 1 and seq 2 tube.

Red was observed for the seq 9 tube at 25 °C → Failure of the system?

Red was observed for the seq 8 tube at 25°C → Normal results

No pellet was observed for the pSB1C3-mRFP, showing clearly that no bacteria developed

This will force us to incubate in petri dishes rather than liquid temperatures. This is rather grim in a way that our system doesn't work. What is curious is that this system has been characterised in the iGEM and scientific literature, hence the problem might come from our sequences.

Week 4

09/11 - PCR colony sequence 8 (BBa_K2282012) and 9 (BBa_K2282013)

Objective: To check if we have the correct inserts in our colonies from transformations with sequence 8 and 9 on 09/06.

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Transformed colonies (on solid LB medium)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Prepare the PCR mix in each PCR tube (the enzyme is added at the end) stay in ice.

We choose colonies in the dishes:

For Sequence 8: test on 3 white colonies and 3 coloured colonies
For Sequence 9: test on 3 white colonies and 3 coloured colonies
Control: colony containing pSB1C3-amilCP (blue colony)

Total = 13 PCR tubes of 25µL

Each chosen colony has been annotated on the Petri dishes so we will be able to use them again for liquid culture later. They are now stocked in the fridge, protected by parafilm.

Cell membrane destruction:

In each PCR tube: put 8µL of dH20. Pick a colony (with a toothpick or a micropipette tip), add a stab of it into the PCR tube.

Incubate the tubes at 95°C for 5min.

Prepare the PCR mix according to the following informations:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	7µL	1X
10mM dNTPs	3,5µL	200µM (1/50)
100µM Prefix Primer	3,5µL	1µM
100µM Suffix Primer	3,5µL	1µM
Taq DNA Polymerase	3,5µL	2,5 units/50µL PCR
Nuclease-free water	172,25µL	qs 350µL

Calculation of H2O: 8 x 13 = 104µL (the quantity of water already added separately in the tubes) ; 276,25 - 104 = 172,25µL

Put 17µL of mix in each tube (25 - 8 = 17).

Cycles for the thermocycler:

Sequence 8:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Sequence 9:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	2 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

We make an agarose gel (cf our Protocols).

Deposit plan:

Ladder
Sequence 8 - Colony 1 (white) 37°C
Sequence 8 - Colony 2 (white) 37°C
Sequence 8 - Colony 3 (white) 37°C
Sequence 8 - Colony 4 (Red) 37°C
Sequence 8 - Colony 5 (Red) 37°C
Sequence 8 - Colony 6 (Red) 37°C
Sequence 9 - Colony 1 (white)
Sequence 9 - Colony 2 (white) 37°C
Sequence 9 - Colony 3 (white)
Sequence 9 - Colony 4 (Red) 30°C
Sequence 9 - Colony 5 (Red) 30°C
Sequence 9 - Colony 6 (Red) 37°C
Control (Blue)
Ladder

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Waited results:

Sequence 8, we are waiting for a band at 1202bp.
Sequence 9: 2076bp.
Control (amilCP): 669bp.

Results:

Interpretation:

37°C - Sequence 8 (white colonies): unexpected bands at 2000pb

→ sequence 9? amilCP not expressed? mRFP (wrong backbone?)

37°C - Sequence 8 (red colonies): expected bands at 1000pb

→ should be sequence 8

Sequence 9 (white colonies): We obtained two bands with the expected size (2000 pb)

30 & 37°C - Sequence 9 (red colonies): unexpected bands at 1000pb

→ Results are not viable and we can not make interpretations. We need to perform this experiment again.

09/11 - PCR on sequences 8 and 9

Objective: To amplify sequences 8 and 9.

Material:

DNA template (seq 8 BBa_K2282012 and seq 9 BBa_K2282013)
10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature.

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	1.5 µL	13.5 µL	500 U/mL
Primers (100µM)	1.5 µL	13.5 µL	10 µM

We prepared 2 tubes of 25µL, one for the sequence 8 and one for the sequence 9, according to the following instructions:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	2,5µL	1X
1mM dNTPs	0,5µL	200µM (1/50)
10µM Prefix Primer	2,5µL	1µM
10µM Suffix Primer	2,5µL	1µM
Taq DNA Polymerase 500U/mL	,5µL	1,25 units/25µL PCR
DNA template (10ng/µL)	1µL
Nuclease-free water	13,5µL	qs 25µL

PCR program on the thermocycler:

Sequence 8 and control pSB1C3-mRFP:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Sequence 9:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	2 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

09/11 - PCR on sequence 1 (BBa_K2282005)

Objective: To amplify our sequence 1 (BBa_K2282005) to further ligate it into pSB1C3-mRFP.

Material:

DNA template (seq 1 BBa_K2282005)
10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Sequence 1

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	1 µL	9 µL	500 U/mL
Primers (100µM)	1 µL	9 µL	10 µM

We prepared a 50µL PCR mix volume according to the following instructions:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	5µL	1X
10mM dNTPs	1µL	200µM (1/50)
100µM Prefix Primer	5µL	1µM
100µM Suffix Primer	5µL	1µM
Taq DNA Polymerase 500U/mL	5µL	2,5 units/50µL PCR
DNA template (10ng/µL)	2µL
Nuclease-free water	27µL	qs 50µL

The PCR mix was divided in two tubes of 25µL each.

PCR program on the thermocycler:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

The two tubes of sequence 1 will be mixed together

09/11 - Digestion pSB1C3-seq2

Objective: To digest a plasmid pSB1C3-Seq 2 (amilCP + DS box) taken from a miniprep of 08/30.

Material:

Plasmid pSB1C3-seq2
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepare the mix for the digestion of pSB1C3-Seq 2 according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
PstI	1µL	40U/µL
pSB1C3-Seq 2	10µL	~10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	33µL

We proceeded 2h at 37°C and heat inactivated for 20 min at 80°C.

For the migration, we are going to use 2/4/8µL of the digestion product to quantify the backbone concentration efficiently.

09/11 - Digestion of pSB1C3-mRFP

Objective: To digest our plasmid pSB1C3-mRFP from a miniprep of the 08/14.

Material:

Plasmid pSB1C3-mRFP (BBa_J04450 from IGEM Kit)
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepared the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
PstI	1µL	40U/µL
pSB1C3-mRFP	10µL	10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	33µL

We incubated 2h at 37°C.

Heat inactivation was performed at 80°C for 20 min.

For the migration, we are going to use 2, 4, 8µL of the digestion product to quantify the backbone concentration efficiently.

09/12 - Migration of PCR and digestions products

Objective: To perform to gel migration of the products of PCR for sequences 1 (BBa_K2282005), 8 (BBa_K2282012) and 9 (BBa_K2282013), and the products of digestions of pSB1C3-mRFP and pSB1C3-seq2.

Material:

Agarose
Distilled water
TAE buffer 50X
Gel cast and combs
Electrophoresis cuve
Microwave
Micropipettes and tips
2-Log DNA Ladder (0.1-10.0 kb) (NEB)
Gel loading dye purple 6X (NEB)

Protocol:

We made an agarose gel (cf our Protocols section)

We prepared the mixes:

For sequences 1, 8, 9: 7µL PCR product + 1,4µL 6X loading dye → 8,4µL total
For digestion products: 15µL digestion product + 3µL 6X loading dye → 18 µL total

Plan of deposit:

Ladder (5µL)
seq 1 - 2µL DNA (2,4µL of the mix)
seq 1 - 4µL DNA (4,8µL of the mix)
pSB1C3-mRFP - 2µL DNA (2,4µL of the mix)
pSB1C3-mRFP - 4µL DNA (4,8µL of the mix)
pSB1C3-mRFP - 8µL DNA (9,6µL of the mix)
seq 8 - 2µL DNA (2,4µL of the mix)
seq 8 - 4µL DNA (4,8µL of the mix)
seq 9 - 2µL DNA (2,4µL of the mix)
seq 9 - 4µL DNA (4,8µL of the mix)
pSB1C3-seq2 - 2µL DNA (2,4µL of the mix)
pSB1C3-seq2 - 4µL DNA (4,8µL of the mix)
pSB1C3-seq2 - 8µL DNA (9,6µL of the mix)
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

We observe the waited bands for sequences 1 and 9, and also for the digested plasmids.

However we do not see anything for sequence 8.

We estimated DNA quantity from the gel observation:

Sample	Quantity in the gel	Quantity in the tube	Quantity after PCR clean up (-25%)
Sequence 1	22,5ng in 4µL	242,09ng in 43µL	181,57ng in 50µL → 3,63 ng/µL
Sequence 9	61ng in 4µL	274,5ng in 18µL	205,88ng in 50µL → 4,12 ng/µL
pSB1C3-mRFP digested	28,5ng in 8µL	3,56 ng/µL
pSB1C3-seq 2 digested	50ng in 8µL	6,25 ng/µL

09/12 - PCR sequences 8 (BBa_K2282012) & 9 (BBa_K2282013)

Part 1: PCR

Objective: To amplify sequences 8 and 9.

Material:

DNA template (seq 8 BBa_K2282012 and seq 9 BBa_K2282013)
10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	1 µL	9 µL	500 U/mL
Primers (100µM)	1 µL	9 µL	10 µM

For a 50µL PCR mix volume:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	5µL	1X
1mM dNTPs	1µL	200µM (1/50)
10µM Prefix Primer	5µL	1µM
10µM Suffix Primer	5µL	1µM
Taq DNA Polymerase 500U/mL	5µL	2,5 units/50µL PCR
DNA template (10ng/µL)	2µL
Nuclease-free water	27µL	qs 50µL

PCR mix is divided in two tubes of 25µL each.

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	seq 8: 1 min (30 sec/kb) seq 9: 2 min	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

The two 25µL tubes of sequence 8 and 9 will be mixed together.

Part 2: Gel Migration

Protocol:

Please refer to our Protocols for the agarose gel preparation.

We prepared the mixes as follow:

7µL PCR product + 1,4µL 6X loading dye → 8,4µL total

Deposit plan:

Ladder - 5µl
seq 8 - 2µL DNA (2,4µL of the mix)
seq 8 - 4µL DNA (4,8µL of the mix)
seq 9 - 2µL DNA (2,4µL of the mix)
seq 9 - 4µL DNA (4,8µL of the mix)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

There was nothing on gel, except the Ladder.

Interpretation:

We will need to get back to the PCR of the sequences 9 and 1 made the day before to clean them up and pursue ligations.

09/13 - Digestion 1 (BBa_K2282005) & 9 (BBa_K2282013) + pSB1C3-seq2 & mRFP

Objective: Sequences 1 and 9 were purified by following the PCR clean-up Protocol. We now want to digest them in order to insert them into pSB1C3 backbone.

Material:

Plasmid pSB1C3-mRFP, pSB1C3-seq2 (BBa_K2282006) and seq 1 (BBa_K2282005) and 9 (BBa_K2282013)
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepared the mixes for sequences 1 and 9 according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
PstI	1µL	40U/µL
Purified sequence	30µL	10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	13µL

We prepared the mix for pSB1C3-mRFP according to the following instructions:

NOTE: SpeI WAS MISTAKEN FOR PstI, HENCE THE DIGESTION WAS STILL CARRIED ON BUT ANOTHER DIGESTION OF THE PSB1C3-SEQ2 BACKBONE WAS NEEDED, WITH ECORI AND SPEI THIS TIME.

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
SpeI	1µL	40U/µL
pSB1C3-seq2	10µL	10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	33µL

We also performed a control for the ligation:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
pSB1C3-mRFP	10µL	10ng/µL
10X NEB buffer 2.1	5µL	1X
Ultrapure water	34µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

09/13 - Ligations of sequences 1, 8, 9 in pSB1C3 backbone

Objective: To ligate the sequences in the pSB1C3 backbone.

Material:

Digested insert (Sequences 1, 8 and 9)
Digested backbone (pSB1C3)
10X T4 DNA Ligase Buffer (NEB)
T4 DNA ligase (400000U/mL)
Ultrapure water
Eppendorf tubes
Micropipettes and tips

Protocol:

Here is the concentration for each DNA component we have:

Seq 1: 3,63 ng/µL
Seq 9: 4,12 ng/µL
Backbone from pSB1C3-mRFP: 3,56 ng/µL
Backbone from pSB1C3-seq2: 6,25 ng/µL
Seq 8 (from 09/05): 6,7ng/µL
Seq 9 (from 09/05): 10,1ng/µL

We performed ligations with a 1:5 ratio.

	seq 1	seq 9	seq 8 (09/05)	seq 9 (09/05)	Control ligase +	Control ligase -
insert	11,85 µL	15,3 µL	10,8µL	12,4µL	-	-
backbone	pSB1C3-mRFP 5,25µL	pSB1C3-seq2 2µL	pSB1C3-seq2 4µL	pSB1C3-seq2 4µL	pSB1C3-mRFP (EcoRI) 5µL	pSB1C3-mRFP (EcoRI) 5µL
10X T4 DNA Ligase Buffer	2µL	2µL	2µL	2µL	2µL	2µL
T4 DNA Ligase	1µL	1µL	1µL	1µL	1µL	0
dH2O	0	0	2,2µL	0	12µL	13µL
Total volume	20µL	20µL	20µL	20µL	20µL	20µL

We performed the ligations Overnight in a 16°C room.

09/14 - Transformations with sequences 1, 8, 9

Objective: To insert the ligated sequences into E.coli.

Material:

Ligated plasmids
Competent cells (DH5-alpha)
LB medium liquid and solid
SOC medium
Chloramphenicol
Ice
Eppendorf tubes
Micropipettes and tips
Waterbath
Petri dishes
Glass beads
Incubator
Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Plate	Competent cells	SOC medium
1	20µL	LB + Chloramphenicol	50µL	930µL
8 old	20µL	LB + Chloramphenicol	50µL	930µL
9 old	20µL	LB + Chloramphenicol	50µL	930µL
9	20µL	LB + Chloramphenicol	50µL	930µL
L +	20µL	LB + Chloramphenicol	50µL	930µL
L -	20µL	LB + Chloramphenicol	50µL	930µL
control mRFP	0µL	LB + Chloramphenicol	50µL	90µL
control seq2	0µL	LB + Chloramphenicol	50µL	940µL

tube 1: Sequence 1 in pSB1C3-mRFP
tube 8 (09/05): Sequence 8 digested (from 09/05) in pSB1C3-seq2 (double digested) (25°C & 37°C)
tube 9 (09/05): Sequence 9 digested (from 09/05) in pSB1C3-seq2 (double digested) (25°C & 37°C)
tube 9: Sequence 9 digested (09/13) in pSB1C3-seq2 (double digested) (25°C & 37°C)
tube L+: pSB1C3-mRFP (digested with EcoRI) + ligase
tube L-: pSB1C3-mRFP (digested with EcoRI)
tube control seq2: pSB1C3-seq 2 not digested
tube control mRFP: pSB1C3-mRFP not digested (25°C & 37°C)

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat water bath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the water bath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and 850µL of supernatant was discarded. Bacteria were resuspended into the remaining culture media and bacteria were spread in the corresponding petri dishes.

Results:

A - Controls

B - Transformations with our sequences

NOTE: There is a mistake on the plates, we wrote 30°C instead of 25°C.

Interpretation:

Our controls worked, even if there is maybe too much colonies for the dish without ligase (B-4). However, we obtained only one red colony for sequence 9 at 37°C, and we suppose that blue colonies are the pSB1C3-seq2 plasmid (B-2). We obtained very few colonies at 25°C, all of them are white (B-3 and B-4).

For sequence 8 (from 09/05), we had also only few white colonies at 25°C (B-5). We had also only 2 red colonies the plate (B-6) at 37°C which are supposed to correspond to the pSB1C3-seq 8 plasmid.

For sequence 1, we observed 2 red colonies (B-7), which are supposed to be the pSB1C3-mRFP plasmid.

We will perform PCR colony in order to confirm the presence of the plasmids.

Week 5

09/18 - PCR colony from 09/14 transformations

Objective: To verify if the desired inserts are found in the colonies we obtained on the 09/14 transformations.

Part 1: PCR colony

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Prepare the PCR mix in each PCR tube (the enzyme is added at the end). Manipulate in ice.

We take colonies from the dishes:

Sequence 8, 25°C: 3 white colonies (called 8-1 to 8-3)
Sequence 8, 37°C: 2 red colonies (called 8-4 and 8-5)
Sequence 9, 25°C, OLD: 3 white colonies (called 9-1, 9-2, 9-3)
Sequence 9, 25°C, NEW: 3 white colonies (called 9-4 to 9-6)
Sequence 9, 37, OLD: 1 red colony (called 9-7)
Control: pSB1C3-mRFP

Total = 13 colonies

Each chosen colony has been annotated on the petri dishes so we will be able to use them again for liquid culture later. They are now stocked in the fridge, protected by parafilm.

Cell membrane destruction:

In each PCR tube: put 8µL of ddH2O. Pick a colony (with a toothpick or a micropipette tip), add a stab of it into the PCR tube.

Incubate the tubes at 95°C for 5 min.

Prepare the PCR mix:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	35µL	1X
10mM dNTPs	7µL	200µM (1/50)
100µM Prefix Primer	3,5µL	1µM
100µM Suffix Primer	3,5µL	1µM
Taq DNA Polymerase	3,5µL	2,5 units/50µL PCR
Nuclease-free water	172,25µL	qs 350µL

Put 17µL of mix in each tube (25-8=17µL)

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	seq 8: 1 min (30 sec/kb) seq 9: 2 min	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Gel migration

Material:

Agarose
Distilled water
TAE buffer 50X
Gel cast and combs
Electrophoresis cuve
Microwave
Micropipettes and tips
2-Log DNA Ladder (0.1-10.0 kb) (NEB)
Gel loading dye purple 6X (NEB)

Protocol:

Deposit plan:

Ladder
8-1 (Sequence 8, 25°C)
8-2 (Sequence 8, 25°C)
8-3 (Sequence 8, 25°C)
8-4 (Sequence 8, 37°C)
8-5 (Sequence 8, 37°C)
9-1 (sequence 9, 25°C, from 09/05)
9-2 (sequence 9, 25°C, from 09/05)
9-3 (sequence 9, 25°C, from 09/05)
9-4( sequence 9, 25°C)
9-5 (sequence 9, 25°C)
9-6 (sequence 9, 25°C)
9-7 (sequence 9, 37°C, from 09/05)
Control
Ladder

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Except the Ladder, we do not see any band in the gel.

Interpretation:

We may have made a mistake while performing the PCR colony.

09/18 - Digestion pSB1C3-mRFP

Objective: The goal here is to simply digest our plasmid pSB1C3-mRFP from a miniprep of the 08/02.

Material:

Plasmid pSB1C3-mRFP (BBa_J04450 from IGEM Kit)
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepared the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1µL	40U/µL
PstI	1µL	40U/µL
pSB1C3-mRFP	10µL	10ng/µL
10X NEB buffer 2.1	5µL	41X
Ultrapure water	33µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

For the agarose gel migration, we are going to use 2, 4 and 8µL of the digestion product to quantify the DNA concentration efficiently.

09/20 - PCR seq 1, 7, 8, 9 + gel migration + PCR clean up + digestion + ligation

Part 1: PCR

Objective: To amplify our sequences, then digest and ligate them into our backbone in order to transform bacteria the day after.

Material:

Forward primer (100µM) synthesized by IDT
Reverse primer (100µM) synthesized by IDT
DNA template (seq 1=BBa_K2282005, seq 7=BBa_K2282011, seq 8=BBa_K2282012, seq9=BBa_K2282013)
10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Taq DNA polymerase (5000U/mL)
DNase/RNase free water
PCR tubes
Micropipettes and tips
Thermocycler

Protocol:

Instead of one 50µL reaction mix we prepared two 25µL reaction mix. The PCR is then performed with only 18 cycles to reduce the risk of errors (as Taq polymerase is not very accurate). Finally the two PCR products are mixed together before the clean up.

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	2,2µL	19,8µL	500U/mL
Primers (100µM)	2,2µL	19,8µL	10µM

For a 25µL PCR mix volume:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	2,5µL	1X
1mM dNTPs	0,5µL	200µM (1/50)
10µM Prefix Primer	2,5µL	1µM
10µM Prefix Primer	2,5µL	1µM
10µM Suffix Primer	2,5µL	1µM
Taq DNA Polymerase 500U/mL	2,5µL	2,5 units/50µL PCR
DNA template (10ng/µL)	1µL
Nuclease-free water	13,5µL	qsp 25 µL

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	seq 1, 7, 8: 1 min (30 sec/kb) seq 9: 2 min	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Gel Migration

Material:

Agarose
Distilled water
TAE buffer 50X
Gel cast and combs
Electrophoresis cuve
Microwave
Micropipettes and tips
2-Log DNA Ladder (0.1-10.0 kb) (NEB)
Gel loading dye purple 6X (NEB)

Protocol:

We are going to use 2 and 4µl of the PCR products to quantify the sequences concentrations efficiently, and 4, 8 µL of the digested backbone.

Mixes for deposit:

7µL PCR product + 1,4µL 6X loading dye → 8,4µL total

13µL of digested backbones + 2,6µL of 6X loading buffer → 15,6µL total

Deposit plan:

Ladder - 5µL
seq 1 - 2µL DNA: 2,4µl of the mix
seq 1 - 4µL DNA: 4,8µL of the mix
seq 7 - 2µL DNA: 2,4µl of the mix
seq 7 - 4µL DNA: 4,8µL of the mix
seq 8 - 2µL DNA: 2,4µl of the mix
seq 8 - 4µL DNA: 4,8µL of the mix
seq 9 - 2µL DNA: 2,4µl of the mix
seq 9 - 4µL DNA: 4,8µL of the mix
pSB1C3-mRFP digested - 4µL DNA: 4,8µL of the mix
pSB1C3-mRFP digested - 8µL DNA: 9,6µL of the mix
pSB1A3-mRFP digested - 4µL DNA: 4,8µL of the mix
pSB1A3-mRFP digested - 8µL DNA: 9,6µL of the mix
pSB1C3-seq 2 digested - 4µL DNA: 4,8µL of the mix
pSB1C3-seq 2 digested - 8µL DNA: 9,6µL of the mix

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

The results are as we expected except for the sequence 9 (to do again).

Quantity estimations:

Ladder - 5µL
Sequence 1: 241,9ng in 43µL
Sequence 7: 107,5ng in 43µL
Sequence 8: 215ng in 43µL
Backbones: 693,8 in 37µL → 18,8 ng/µL final

Part 3: Digestion

Objective: We performed a PCR clean-up for the sequences 1, 7, 8. We now want to digest them prior to perform a ligation.

Material:

Plasmid pSB1C3-mRFP (BBa_J04450), Inserts: seq 1=BBa_K2282005, seq
7=BBa_K2282011, seq 8=BBa_K2282012, seq9=BBa_K2282013
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Material:

We prepared the mixes according to the following instructions:

For sequences 1, 7 and 8:

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
PstI	1 µL	40U/µL
Purified sequence	30 µL	10 ng/µL
10X NEB buffer 2.1	5 µL	1X
Ultrapure water	13 µL

For pSB1C3-mRFP (control ligase):

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
backbone	10 µL	10 ng/µL
10X NEB buffer 2.1	5 µL	1X
Ultrapure water	34 µL

For pSB1A3-mRFP (miniprep from the 08/14), and pSB1C3-seq2 (miniprep from the 08/30):

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	40U/µL
PstI	1 µL	40U/µL
backbone	10 µL	10 ng/µL
10X NEB buffer 2.1	5 µL	1X
Ultrapure water	33 µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

Final concentrations:

Sequence 1: 3,6 ng/µL
Sequence 7: 1,61 ng/µL
Sequence 8: 3,2 ng/µL

Part 4: Ligation

Material:

Digested inserts (see previous digestion)
Digested backbone (see previous digestion)
10X T4 DNA Ligase Buffer (NEB)
T4 DNA ligase (400000U/mL)
Ultrapure water
Eppendorf tubes
Micropipettes and tips

Protocol:

	seq 1 1:3	seq 1 1:5	seq 7 1:3	seq 7 1:5	seq 8 1:3	seq 8 1:5	Control ligase +	Control ligase -
insert	9,6 µL	15,9 µL	17,3 µL	16 µL	13,6 µL	17,2 µL	-	-
backbone	pSB1C3-mRFP (double digested, 25ng) 1,3µL	pSB1C3-mRFP (double digested, 25ng) 1,3µL	pSB1A3-mRFP (double digested, 18ng) 1µL	pSB1A3-mRFP (double digested, 10ng) 1µL	pSB1C3-seq2 (double digested, 25ng) 1,3µL	pSB1C3-seq2 (double digested, 19ng) 1µL	pSB1C3-mRFP (EcoRI) 5µL	pSB1C3-mRFP (EcoRI) 5µL
10X T4 DNA Ligase Buffer	2µL	2µL	2µL	2µL	2µL	2µL	2µL	2µL
T4 DNA Ligase	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL	1 µL	0
dH2O	6,1 µL	0	0	0	2,1 µL	0	12 µL	13 µL
Total volume	20 µL	20,2 µL	21,3 µL	19,5 µL	20 µL	21,2 µL	20 µL	20 µL

Ligation was performed overnight in a 16°C room.

09/21 - Transformations with sequences 1, 7, and 8

Objective: To transform E. coli with the sequences 1, 7, and 8.

Material:

Ligated plasmids (see previous ligation)
Competent cells (DH5-alpha)
LB medium liquid and solid
T4 DNA ligase (400000U/mL)
SOC medium
Chloramphenicol
Eppendorf tubes
Micropipettes and tips
Waterbath
Petri dishes
Glass beads
Incubator
Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Plate	Competent cells	SOC medium
1 (1:3)	20 µL	LB + Chloramphenicol	50µL	930µL
1 (1:5)	20 µL	LB + Chloramphenicol	50µL	930µL
7 (1:3)	20 µL	LB + Ampicilline	50µL	930µL
7 (1:5)	20 µL	LB + Ampicilline	50µL	930µL
8 (1:3)	20 µL	LB + Chloramphenicol	50µL	930µL
8 (1:5)	20 µL	LB + Chloramphenicol	50µL	930µL
Ligase +	20 µL	LB + Chloramphenicol	50µL	930µL
Ligase -	20 µL	LB + Chloramphenicol	50µL	930µL
pSB1A3-mRFP	10 µL	LB + Ampicilline	50µL	990µL
pSB1C3-seq2	10 µL	LB + Chloramphenicol	50µL	990µL

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL!

Add qs 950 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and 850µL of supernatant was discarded. Bacteria were resuspended into the remaining culture media and bacteria were spread in the corresponding petri dishes.

Results:

Interpretation:

All the transformations worked with coherent results. We can indeed see the huge bacterial concentration in miniprep transformed bacteria (as the DNA is very concentrated) and logic results for the control (more colonies with ligase-ligated plasmid than with naturally ligated plasmid only). We are going to use the color reporters to select the appropriate colonies when necessary. Note that the molar ratio didn’t seem to impact the transformation results.

09/21 - PCR sequence 9 (BBa_K2282013)

Objective: As we seems to have had trouble to amplify sequence 9 before, we want to succeed this time! We will use 2 different type of DNA polymerase in order to compare their efficiency.

Material:

Forward primer (100µM) synthesized by IDT
Reverse primer (100µM) synthesized by IDT
DNA template(BBa_K2282013)
10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Taq DNA polymerase (5000U/mL)
DNase/RNase free water
PCR tubes
Micropipettes and tips
Thermocycler

Protocol:

Taq polymerase: We prepared two 25µL reaction mixes. The PCR is then performed with only 18 cycles to reduce the risk of errors (as Taq polymerase is not very accurate compared to Q5). Finally the two PCR products are mixed together before the clean up.

Q5 polymerase: We prepared one 25µL reaction mix with the remaining Q5 polymerase we had in the fridge.

Pre-dilutions (1/10):

	Sample	dH2O DNase/RNase free	Final concentration
Taq Polymerase	2.2 µL	19.8 µL	500 U/mL
Primers (100µM)	2.2 µL	19.8 µL	10 µM

Assemble all reaction components on ice and quickly transfer to a thermocycler preheated to the denaturation temperature (95°C).

Composition of the PCR mix (25µL) for Taq Polymerase:

Components	Volume	Concentration
10X Standard Taq Reaction Buffer	2.5 µL	1X
10mM dNTPs	0.5 µL	200µM (1/50)
10µM Prefix Primer	2.5 µL	1µM
10µM Suffix Primer	2.5 µL	1µM
Taq DNA Polymerase 500U/mL	2.5 µL	2,5 units/50µL PCR
DNA template (10ng/µL)	2 µL
Nuclease-free water	12,5 µL	qsp 25µL

Composition of the PCR mix (25µL) for Q5 polymerase:

Components	Volume	Concentration
10µM Prefix Primer	2.5 µL	1µM
10µM Suffix Primer	2.5 µL	1µM
Q5 polymerase 2X	12.5 µL	1X
DNA template (10ng/µL)	2 µL
Nuclease-free water	5,5 µL	qsp 25µL

PCR thermocycler programs:

-> For Taq polymerase reaction:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	2 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

-> For Q5 polymerase reaction:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	98°C
30	Denaturation	10 sec	98°C
	Annealing	30 sec	60°C
	DNA polymerization	1 min	72°C
0	DNA polymerization	2 min	72°C
	FINISH	∞	4°C

09/23 - PCR colony from 21/09 transformations + culture of pSB1C3-seq 2 transformed bacteria

Part 1: PCR colony

Objective: To check if we have the expected insert in our colonies

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNase/RNase free water
PCR tubes
Micropipettes and tips
Thermocycler

Protocol:

Prepare the PCR mix in each PCR tube (the enzyme is added at the end) stay in ice.

From:

Sequence 1: 3 blue colonies (named 1-1 to 1-3)

Sequence 7: 3 red colonies (7-1 to 7-3) + 3 white colonies (7-4 to 7-6)

Sequence 8: 2 blue colonies (8-1, 8-2), 2 white colonies (8-3, 8-4)

Total = 13

Each chosen colony has been annotated on the Petri dishes so we will be able to use them again for liquid culture later. They are now stocked in the fridge, protected by parafilm.

Cell membrane destruction:

In each PCR tube: put 8µL of dH2O. Pick a colony (with a toothpick or a micropipette tip), add a stab of it into the PCR tube.

Incubate the tubes at 95°C for 5 min.

Prepare the PCR mix:

Components	Volume	Final concentration
10X Standard Taq Reaction Buffer	35 µL	1X
10mM dNTPs	7 µL	200µM (1/50)
10µM Prefix Primer	3.5 µL	1µM
10µM Suffix Primer	3.5 µL	1µM
Taq DNA Polymerase	3.5 µL	2,5 units/50µL PCR
Nuclease-free water	172,25 µL	qs 350µL

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
30	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	2 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Agarose gel migration

Please refer to our Protocol to make an agarose gel in our Protocol section.

Preparation of the samples to load in the gel:

5µL of the PCR mix + 1µL of 6X Loading Dye

Deposit plan:

Ladder (5µL)
1-1 (blue)
1-2 (blue)
1-3 (blue)
7-1 (red)
7-2 (red)
7-3 (red)
7-4 (white)
7-5 (white)
7-6 (white)
8-1 (blue)*
8-2 (blue)*
8-3 (white)
8-4 (white)
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

Sequence 1: We have the correct insert for blue colonies

Sequence 7: We have the correct insert with white colonies

*Sequence 8: We made a mistake, we took blue colonies instead of red colonies. The correct insert is supposed to be in the red colonies.

Part 3: Culture (on 09/25)

2 blue colonies with seq 1 (LB + Chloramphenicol)
2 white colonies with seq 7 (LB + Ampicillin)
2 red colonies with seq 8 (LB + Chloramphenicol) incubation at 37°C.

Also, we put a transformed colony with pSB1C3-seq 2 in culture (in LB+chloramphenicol), at 37°C.

Week 6

09/25 - Miniprep of pSB1C3-seq 9, pSB1C3-seq 2 and pSB1A3-mRFP

Objective: We want to isolate the plasmid constructions containing sequences 2 and 9.

Part 1: Miniprep

Material:

Monarch® Plasmid Miniprep Kit (NEB)
Micropipettes + tips
Eppendorf tubes
Centrifuge
Ice

Protocol:

Details of the tubes:

Plasmid	Date of transformation	Conditions
pSB1C3-Seq 9	09/21	LB medium + Chloramphenicol - Not red when taken off the incubator but became red during the manipulation
pSB1C3-Seq 2	09/23	LB medium + Chloramphenicol
pSB1A3-mRFP	09/23	LB medium + Ampicillin

The Monarch protocol and Antoine's indications were used:

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.

Store Plasmid Neutralization Buffer (B3) at 4°C after opening.

Note:unlike other commercial kits, all wash steps are required.

1) Pellet 1–5 ml (3 mL is fine) bacterial culture (3ml for this experiment) by centrifugation for 30 seconds (we prefered to centrifuge 1min because of the high cell concentration). Discard supernatant.

2) Resuspend pellet in 200 µl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.

3) Lyse cells by adding 200 µl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute at room temperature.

Note: Care should be taken not to handle the sample roughly and risk shearing chromosomal DNA, which will co-purify as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.

4) Neutralize the lysate by adding 400 µl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.

Note: Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.

5) Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.

Note: Spin time should not be less than 2 minutes. Careful handling of the tube will ensure no debris is transferred and the 2 minute recommended spin can be successfully employed to save valuable time. For culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.

To save time, spin for two minutes only.

6) Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.

To save time, spin for 30 seconds, instead of 1 minute.

If using a vacuum manifold* instead of centrifugation, insert the column into a manifold and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

7) Re-insert column in the collection tube and add 200 µl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.

Note: The collection tube is designed to hold 800 µl of flow-through fluid and still allow the tip of the column to be safely above the top of the liquid. Empty the tube whenever necessary to ensure the column tip and flow-though do not make contact.

To save time, spin for 30 seconds, instead of 1 minute.

If using a vacuum manifold, add 200 µl of Plasmid Wash Buffer 1 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

* Make sure to follow the manifold manufacturer's instructions to set-up the manifold and connect it properly to a vacuum source.

8) Add 400 µl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

When using a manifold add 400 µl of Plasmid Wash Buffer 2 and switch the vacuum on. Allow the solution to pass through the column, then switch the vacuum source off.

9) Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.

If using a vacuum manifold: Since vacuum set-ups can vary, a 1 minute centrifugation is recommended prior to elution to ensure that no traces of salt and ethanol are carried over to the next step.

10) (repeat this step two times to get more plasmids, total volume=60ul) Add ≥ 30 µl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Delivery of the Monarch DNA Elution Buffer should be made directly to the center of the column to ensure the matrix is completely covered for maximal efficiency of elution. Additionally, yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated as a result of dilution. For larger plasmids (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to eluting and extending the incubation time after buffer addition to 5 minutes can improve yield.

Part 2: Gel Migration

Material and Protocol:

Please refer to our Protocol section for the agarose gel fabrication.

We prepared the samples to load in the gel:

Minipreps: 5µL sample + 1µL 6X loading dye

PCR samples of 09/21: 7µL PCR product + 1,4µL 6X loading dye --> 8,4µL total

Quantity of DNA to load:

2µl: 2,4µl of the mix

4µl: 4,8µl of the mix

Deposit plan:

Ladder (5µL)
pSB1A3-mRFP (miniprep) 5µL
Seq9 (miniprep) 5µL
pSB1C3-Seq2 (miniprep) 5µL
PCR Q5 2,4µL
PCR Q5 4,8 µL
PCR Taq 2,4µL
PCR Taq 4,8µL

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

Minipreps: There are problems with all the sizes we observed. They are unexpected. Maybe we should send the samples to be sequenced by GATC to be sure.

Sequence 9: There is still nothing. We definitely have a problem to amplify this particular sequence.

09/26 - Miniprep on sequences 9 (09/08), 8 (09/08) and 1 (09/25)

Objective: We want to isolate the plasmid constructions containing sequences 1, 8 and 9.

Material:

Monarch® Plasmid Miniprep Kit (NEB)
Micropipettes + tips
Eppendorf tubes
Centrifuge
Ice

Protocol:

Volume of LB medium in sequence 7 tube (09/25) was too low (5mL), we added 10mL of LB medium containing ampicillin.

Details of the tubes:

pSB1C3-seq 9 08/09 LB medium + Chloramphenicol - red when taken off the incubator )
pSB1C3-seq 8 08/09 LB medium + Chloramphenicol
pSB1C3-seq 1 25/09 LB medium + Chloramphenicol

NB: Pay attention to the elution volume. In order to prepare sample for GATC, we eluted in 60µL nuclease-free water and not 30µL. Indeed, we had approximately 120ng/5µL DNA on electrophoresis gel and GATC asks for 20µL with 30-100ng/µL, therefore our DNA is too much concentrated.

The Monarch protocol and Antoine's indications were used as usual (please refer to our “Protocols” section)

09/27 - Digestions of plasmides and gel migration

Objective:We want to digest the following plasmids with EcoRI-HF and PstI, and then verify by gel migration the efficiency of the digestion step:

pSB1A3-mRFP (23/09)
pSB1C3-seq2 (?)
pSB1C3-seq9 (21/09)
pSB1C3-seq8 (from miniprep of 08/09)
pSB1C3-seq9 (from miniprep of 08/09)
pSB1C3-seq1 (from miniprep of 25/09 culture)
pSB1C3-seq7 (from miniprep of 25/09 culture)
pSB1C3-seq8 (from miniprep of 25/09 culture)

Part 1: Digestion

Material:

Plasmids (Cf above)
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepared the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF	1 µL	80U/µL
PstI	1 µL	80U/µL
Backbone	5 µL	10ng/µL
10X NEB buffer 2.1	2,5 µL	1X
Ultrapure water	15,5 µL	td>

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

Part 2: Gel Migration

Material and Protocol:

We made an agarose gel according to the protocol (please refer to our "Protocols" section).

We prepared the samples for the migration: 5µL of digestion product + 1µL 6X Loading dye

Deposit plan:

pSB1A3-mRFP (23/09)
pSB1C3-seq2 (?)
pSB1C3-seq9 (21/09)
pSB1C3-seq8 (from miniprep of 08/09)
pSB1C3-seq9 (from miniprep of 08/09)
pSB1C3-seq1 (from miniprep of 25/09 culture)
pSB1C3-seq7 (from miniprep of 25/09 culture)
pSB1C3-seq8 (from miniprep of 25/09 culture)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

pSB1A3-mRFP: we are waiting for a band at 2 155 bp (backbone) + a band at 706bp (mRFP). We can't see very well the mRFP band.

pSB1C3-seq 2: we are waiting for a band at 2 070 bp (backbone) + a band at 965 bp (seq 2). It seems to be ok !

pSB1C3-seq 9: we are waiting for a band at 2 070 bp (backbone) + a band at 2076bp (seq 9). We have only a band at 1200bp for the seq 9 for both wells 3 and 5. This length represents only the half of the sequence.

pSB1C3-seq8: we are waiting for a band at 2 070 bp (backbone) + a band at 1202bp (seq 8). It seems to be ok !

pSB1C3-seq1: we are waiting for a band at 2 070 bp (backbone) + a band at 950bp (seq 1). It seems to be ok !

pSB1C3-seq7: we are waiting for a band at 2 070 bp (backbone) + a band at 1110 bp (seq 7). It seems to be ok !

09/29 - Transformation with japanese plasmids (pHeat & pCold) from team Tokyo Tech

Objective: We received the plasmids from the team Tokyo Tech (pCOLD: BBa_K1949000). We need to transform E.coli with them.

Material:

Ligated plasmid
Competent cells (DH5-alpha)
LB medium liquid and solid
SOC medium
Chloramphenicol
Ice
Eppendorf tubes
Micropipettes and tips
Waterbath
Petri dishes
Glass beads
Incubator
Centrifuge

Protocol:

The same protocol as usual was used. Please refers to our “Protocols” section.

Tubes	DNA	Plate	ompetent cells	SOC medium
pCold	10 µL	LB + Chloramphenicol	50µL	940µL
pHeat	10 µL	LB + Chloramphenicol	50µL	940µL
pSB1C3-mRFP	10 µL	LB + Chloramphenicol	50µL	940µL

Results:

Results are observed after 3 days at 37°C.

Interpretation:

Transformation worked for all the sequences, we tried to streak to obtain isolated colonies and it worked. pCOLD seems to be fluorescent but this is very hard to tell since the plate itself produces a blue light. pCOLD should not be fluorescent at 37°C anyway so we are going to assess if it is the case in further characterisations.

09/29 - Sequencing by GATC and results

We sent some of our sequences and our primers to GATC in order to obtain their sequence.

miniprep pSB1C3-seq 1
pSB1C3-seq2
pSB1C3-seq7
miniprep pSB1C3-seq8 (from culture 08/09)
miniprep pSB1C3-seq8 (from culture 25/09)
miniprep pSB1C3-seq9 (from culture 08/09)
pSB1C3-seq9 (from 21/09)

Primer preparation: 2µL primer + 18µL dH2O

Sample preparation: 10µL sample + 10µL dH2O (concentration is estimated between 30 and 50ng/µL)

Results:

1. a) Sequencing of miniprep pSB1C3- seq 1 with primer prefix:

The green part means that we have 100% of identity. For this sequence, we have:

VF2: not covered
Prefix: not covered
the promoter isn't completely covered but it is normal because it is the beginning.
RBS: covered
AmilCP: covered
double terminator: covered
suffix: quite covered (normal: it is the end)
VR: quite covered (normal: it is the end)

This sequencing is good, we have the correct sequence.

1. b) Sequencing of miniprep pSB1C3- seq 1 with primer suffix:

VF2: not covered (normal, it is the end)
Prefix: not covered (normal, it is the end)
promoter: not covered
RBS: not covered
AmilCP: not completely covered
double terminator: quite covered but some mispairing
suffix: quite covered but some mispairing
VR: quite covered but some mispairing

This sequencing is not a very good quality. But we have good results on the previous one so it is OK.

2. a) sequencing of pSB1C3-seq2 with primer prefix:

NO result; either a problem of primer or a problem of sequencing.

2. b) sequencing of pSB1C3-seq2 with primer suffix:

VF2: not covered
Prefix: not covered
promoter: not covered
RBS: not covered
AmilCP: not completely covered (only the end)
double terminator: not completely covered (only the beginning)
suffix: not covered
VR: not covered

We don't have the sequence of AmilCP/DSB. This is a problem of sequencing or primer. We cannot take these results into account.

3. a) sequencing of pSB1C3-seq7 with primer prefix:

VF2: not covered (normal it is the beginning)
Prefix: not covered (normal it is the beginning)
UPelmt/CspA prom: not completely covered, only the end.
5'UTR: OK
AmilCP/DSB: OK
double terminator: not completely covered
suffix: not covered
VR: not covered

This sequencing result is quite good as we can see the main parts: Upelmt/CSPAprom (not entirely), 5'UTR, AmilCP/DSB.

3. b) sequencing of pSB1C3-seq7 with primer suffix:

Not a good sequencing. We have only the double terminator. But we can use the previous one which is good.

4. a) sequencing of miniprep pSB1C3-seq8 (culture 08/09) with primer prefix:

pL promoter: not completely covered, there are some mismatches
eRFP: OK

This sequencing result is acceptable but the pL promoter is not completely sequenced.

4. b) sequencing of miniprep pSB1C3-seq8 (culture 08/09) with primer suffix:

Not very relevant: we have only the double terminator. But we can use the previous result.

5) a. sequencing miniprep pSB1C3-seq8 (culture 25/09) with primer prefix:

NO RESULT

5) b. sequencing miniprep pSB1C3-seq8 (culture 25/09) with primer suffix:

NO RESULT

6) a. sequencing miniprep pSB1C3-seq9 (culture 08/09) with primer prefix:

VF2: OK
Prefix: OK
promoter: OK
RBS: quite OK
CI857: quite OK
Terminator: not completely covered.
eColi operon: not covered
eRFP: not covered
RBS: not covered
pL: not covered

We don't have the entire sequence. (remember the gel: we had only the half of the sequence).

6) b. sequencing miniprep pSB1C3-seq9 (culture 08/09) with primer suffix:

VF2: not covered
Prefix: not covered
promoter: not covered
RBS: not covered
CI857: not covered
Terminator: not covered.
eColi operon: OK
eRFP: not covered
RBS: not covered
pL: not covered

Here we have the end of the sequence. On the previous one, we have the beginning. It seems that despite what we saw on the gel yesterday, we have the entire sequence in our plasmid.

7. a) sequencing of pSB1C3-seq9 (21/09) with primer prefix:

VF2: OK
Prefix: OK
promoter: OK
RBS: OK
CI857: Ok but some mismatches
Terminator: OK
eColi operon: not covered
eRFP: not covered
RBS: not covered
pL: not covered

7. b) sequencing of pSB1C3-seq9 (21/09) with primer suffix:

VF2: not covered
Prefix: not covered
promoter: not covered
RBS: not covered
CI857: not covered
Terminator: only the end is covered
eColi operon: OK
eRFP: OK
RBS: OK
pL: OK but not completely covered

Week 7

10/02 - Miniprep for sequences 1 / 2 / 7 / 8 / 9 and cultures for sequences 1 / 2 / 7 / 8 / 9

Objective: To make stocks of our sequenced plasmids and prepare cultures for further characterisation.

Part 1: Minipreps

Material:

Monarch® Plasmid Miniprep Kit (NEB)
Micropipettes + tips
Eppendorf tubes
Centrifuge
Ice

Protocol:

The Monarch protocol and Antoine's indications were used. The same protocol as usual was used. Please refers to our “Protocols” section.

Part 2: Cultures of DH5alpha transformed with sequences 1, 2, 7, 8 and 9

Material:

LB medium
Chloramphenicol
50mL falcon tubes
Micropipettes + tips
Incubator

Protocol:

We prepared for each culture 100µL of pre-cultures in 10ml of LB medium with antibiotics.

Remark:

seq8 (1) and seq9(1) → come from culture prepared the 08/09
seq8 (2) and seq(2) → come from culture prepared the 25/09 and 21/09 respectively

10/03 - PCR colony on japanese sequences

Objective: To check if the bacteria have the correct insert sizes.

Part 1: PCR colony

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

Prepare the PCR mix in each PCR tube (the enzyme is added at the end) stay in ice.

Japanese pHeat: 3 colonies (white) (1694bp)

Japanese pCold: 3 colonies (white) (1033bp)

Each chosen colony has been annotated on the petri dishes so we will be able to use them again for liquid culture later. They are now stocked in the fridge, protected by parafilm.

Cell membrane destruction:

In each PCR tube: put 8µL of ddH20. Pick a colony (with a toothpick or a micropipette tip), add a stab of it into the PCR tube. Incubate the tubes at 95°C for 5min.

Prepare the PCR mix:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	17,5 µL	1X
10mM dNTPs	3,8 µL	200µM (1/50)
10µM Prefix Primer	1,75 µL	1µM
10µM Suffix Primer	1,75 µL	1µM
Taq DNA Polymerase	1,75 µL	2,5 units/50µL PCR
Nuclease-free water	148,75µL	qsp 175µL

Put 17µL of mix in each tube (25-8=17)

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
30	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	1 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Gel migration:

Material and Protocol:

We made an agarose gel according to the protocol (please refer to our "Protocols" section).

We prepared the samples to load in the gel:

10µL of the PCR mix + 2µL of 6X loading dye

Deposit plan:

Ladder (5µL)
pCold 1
pCold 2
pCold 3
pHeat 1
pHeat 2
pHeat 3
PCR 9 (old)
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

The gel was unreadable (blurred ladder and GelRed everywhere).

10/03 - OD measure for sequences 1 and 2, and protein extraction

Objective: To quantify a protein expression.

Part 1: OD600nm of bacterial culture (seq 1&2)

Material:

LB medium
Chloramphenicol
Micropipettes + tips
Spectrophotometer
Cuvettes

Protocol:

Blanc = LB + chloramphenicol

OD600nm DH5alpha-pSB1C3-seq1 = 0,719

OD600nm DH5alpha-pSB1C3-seq2 = 0,692

Part 2: Protein extraction

Material:

LB medium
Chloramphenicol
50mL Falcon tubes
Eppendorf tubes
Centrifuge
Spectrophotometer
Cuvettes
Vortex
Freezer
Waterbath

Protocol:

Extraction of total proteins from:

WT
DH5alpha-pSB1C3-seq1
DH5alpha-pSB1C3-seq2

We have to adapt the protocol because the bacterial culture's OD has been measured into LB medium+chloramphenicol. So if we resuspend the proteins into water, we won't be able to compare the OD. So we need to resuspend the proteins into LB medium + chloramphenicol.

Grow the cells and induce protein expression
Take as 1mL sample from the cell culture at OD600 of 1 as experiments you would like to carry out. If the OD of the culture is not 1 you have to correct the sample volume to obtain an equivalent size pellet.
Spin down the cells for 5min at 6000 rpm in a microfuge.
For each pellet, remove supernatant and add 150µL of LB medium + chloramphenicol.
Vortex to resuspend the cells.
Freeze quickly at -20°C and leave for 3 min.
Thaw immediately at 42°C 3mn. Vortex vigorously to mix well.
Repeat the two previous steps for three more times (4 freeze-thaw-vortex cycles in all).
Spin down the cells for 5min at 6000 rpm in a microfuge. Proteins are contained in the supernatant.

Results:

Interpretation:

The protein extraction did not work (the color is still in the pellet).

We will try again the same protocol but with ultrapure water instead of LB, because maybe water is absolutely needed for cell lysis.

10/04 - Protein extraction: AmilCP from sequences 1 and 2

Objective: We want to extract the AmilCP from colonies transformed with AmilCP (seq 1) and with AmilCP + DS box (seq 2).

Material:

Bacterial cultures
Ultrapure water
Eppendorf tubes
Centrifuge
Spectrophotometer
Cuvettes
Vortex
Freezer
Waterbath

Protocol:

Put 1mL of culture into eppendorf tubes
Centrifuge at 6000rpm for 5min
Discard supernatant, resuspend in ultrapure water
Centrifuge again at 6000rpm for 5min, discard supernatant
Freeze-thaw cycles: 5min -20°C; 5min 80°C (4 times)
Resuspend in ultrapure water
Centrifuge at 6000rpm for 5min

Results:

We failed at extracting the proteins.

Week 8

10/09 - Characterisation Protocole 0: Testing the kinetic of evolution of E.coli in Liquid LB at 15°C and 37°C

Objective: To perform a growth curve of E.coli DH5alpha at 37°C and at 15°C to compare the kinetic, as we want to anticipate the kinetic of growth of an E.coli population at 15°C and compare it to the one at 37°C.

Material:

Liquid LB without antibiotic
DH5 alpha E.Coli WT
Sterile Erlenmeyers
Shaker-incubator 37°C 250 rpm and Multitron 15°C 250 rpm
Centrifuge
Spectrophotometer
Cuvettes

Protocol:

Put 50 ml of LB in each erlenmeyers
Incubate 1 ml of E.Coli DH5 alpha WT in the two different erlenmeyers.
Immediately take off 1ml of culture IN ICE and calculate OD600 for t=0
Put one erlenmeyer at 37°C 250 rpm and the other one at 15°C and repeat every 1h30 for each erlenmeyers and note OD600.
Carry on until you reach stationary phase

Results:

Figure 1 - OD measured at 600nm at 15°C and 37°C at different times.

Figure 2 - Evolution of the OD600nm measured according to the time

Interpretation:

At 37°C, exponential phase is reached after 3h and stationary phase after 6h of incubation.

No growth at 15°C.

10/09 - Characterisation Protocole 1: Protein extraction

Objective: To extract the total intracellular proteins from E.coli to measure the absorbance and deduce the proteins (amilCP, mRFP) concentrations.

Material:

SDS 1%
PBS 10X w/o Ca2+ and Mg2+ → PBS 2014 (dubious)
ICE COLD Acetone
Bacteria culture you wish to work on --> culture seq8 (the blue one)
Centrifuge
Eppendorf tubes
Micropipette + tips

Protocol:

Stock: SDS (20%) stock solution

Preparation of a 1% SDS solution: put 10ml of 20% SDS in a 250mL bottle and add 190ml of deionised water.

Preparation of a 1X PBS solution: put 100ml of 10X PBS solution and complete to 1L with deionised water.

1) Cells from 3 ml of culture were harvested by centrifugation (13000 x g 1 min), washed twice with phosphate-buffered saline without Mg2+ and Ca2+ (3), and recentrifuged.

2) The cells were then suspended in 1,5 ml of ice-cold acetone (analytical grade), allowed to stand on ice for 5 min, and collected by centrifugation (7,000 x g).

Experiments demonstrated that acetone treatment from 5 to 30 min or the use of more acetone did not change the efficiency of protein extraction.

3) The proteins were then extracted by incubating with 150ul ml of 1% sodium dodecyl sulfate (SDS) for 2 min.

The acetone pretreatment was found necessary since the direct utilization of detergents did not extract cellular proteins from gram-positive bacteria.

4) To perform an absorption spectrum we completed the volume with 1,5ml of water.

Source: Bhaduri et al., “Simple and Rapid Method for Disruption of Bacteria for Protein Studies”, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1983, p. 941-943

Results:

Interpretation:

Partial results, not all the proteins were extracted but it allows to realise an absorption spectrum. We can see the absorption peak of amilCP and another peak which could be mRFP. (the culture was purple → mix of blue and red).

10/09 - Miniprep for sequences 1 / 2 / 7 / 8 / 9 and cultures for sequences 1 / 2 / 7 / 8 / 9

Objective: To make stocks of our sequenced plasmids and prepare cultures for further characterisation.

Part 1: Miniprep

Material:

Monarch® Plasmid Miniprep Kit (NEB)
Micropipettes + tips
Eppendorf tubes
Centrifuge
Ice

Protocol:

The Monarch protocol and Antoine's indications were used. The same protocol as usual was used. Please refers to our “Protocols” section.

Part 2: Cultures of DH5alpha transformed with seq 1,2,7,8,9

Put 100µL of precultures in 10ml of LB medium with antibiotics.

10/10 - Characterisation Protocol 1: Protein extraction for mRFP

Objective: To extract the total intracellular mRFP from E.coli in order to measure the absorbance and deduce the proteins concentration.

Material:

SDS 1%
PBS 10X w/o Ca2+ and Mg2+ → PBS 2014 (dubious)
ICE COLD Acetone
Bacteria culture you wish to work on → culture seq 8 (the blue one)

Protocol:

Stock: SDS (20%) stock solution

Preparation of a 1% SDS solution: put 10ml of 20% SDS in a 250mL bottle and add 190ml of deionised water.

Preparation of a 1X PBS solution: put 100ml of 10X PBS solution and complete to 1L with deionised water.

1) Cells from 3 ml of culture were harvested by centrifugation (13000 rpm 1 min), washed twice with 1,5 ml of phosphate-buffered saline without Mg2+ and Ca2+ (3) (homogenize with pipeting and vortex), and recentrifuged (13000 rpm, 1min).

2) The cells were then suspended in 1,5 ml of ice-cold acetone (analytical grade), allowed to stand in fridge for 5 min, and collected by centrifugation (13000 rpm 1min).

Tips: Break the pellet with your P1000 tips right after you put the acetone.

Experiments demonstrated that acetone treatment from 5 to 30 min or the use of more acetone did not change the efficiency of protein extraction.

3) The proteins were then extracted by incubating with 150ul ml of 1% sodium dodecyl sulfate (SDS) for 2 min. Homogenize by pipeting and vortexing. Centrifuge 1min at 13000rpm to get the supernatant.

The acetone pretreatment was found necessary since the direct utilization of detergents did not extract cellular proteins from gram-positive bacteria.

4) Put the supernatant in an eppendorf tube and add 1,35ml of water (Vtotal=1,5ml). Vortex. Perform the absorption spectrum. For the blank: 150ul of SDS and 1350µL of water (homogenize).

Source: Bhaduri et al., “Simple and Rapid Method for Disruption of Bacteria for Protein Studies”, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1983, p. 941-943

Results:

The culture used for extraction was seq 9 (1) (we couldn't see the red colour directly but after centrifugation the pellet was slightly pink). A little peak was observed at 584 nm.

10/10 - Characterisation Protocole 2: DSBox effect on AmilCP Properties - Determination of the absorption peak

Objective: To assess the effect of the DSBox alone on amilCP coloration properties (absorbance spectrum).

Material:

O/N culture of Seq 1 at 15 or 37°C
O/N culture of Seq 2 at 15 or 37°C
O/N culture of WT at 15 or 37°C
SDS 1%
PBS w/o Ca2+ and Mg2+
ICE COLD Acetone

Protocol:

1) IN ICE Take 4,5 ml of each culture and use 1,5ml to measure the OD600.

2) IN ICE Note and adjust the OD600 to obtain the same cell concentration in each tubes

3) Once the concentration is adjusted, take 3ml of each adjusted culture and proceed the protein extraction protocol.

4) With the 1,5ml of protein extract of each culture and the blank, make an absorption spectrum and note the results.

You should at the end obtain the complete spectrum of AmilCP both native and modified at both 15 and 37°C

→ Determine the maximal absorbance and the corresponding concentration

NOTE: The protocol failed and the proteins were not extracted. We had to re-do it and include the acetone step again with what was remaining of the samples (600 µl).

This will explain the low absorbance values we obtain in the results. The extraction worked with the acetone.

Results:

After doing the blank with the extracted proteins of the Wild-Type culture, we obtained:

The absorption spectrum of the native AmilCP from the sequence 1:

The absorption spectrum of the modified AmilCP with the DSBox from the sequence 2:

Interpretation:

The peaks are very similar but present some variations, although the native AmilCP spectrum is analysable on a different scale (ymax = 0.002) that the one of the modified AmilCP (y = 0,09) and the resolution of the other smaller peaks is hence different and they are more easily distinguishable. This proves however that the maximum of absorption from the native and the modified AmilCP is the same, at 588 nm. We will carry on the same experiment soon too see if we have similar results.

10/10 - Migration PCR colony japanese plasmids

Objective: To migrate the PCR colony results from the 10/03.

Protocol:

We made an agarose gel according to the protocol (please refer to our "Protocols" section).

Preparation of the samples:

for pCold and pHeat sequences: 10µL PCR + 2µL loading dye
for sequence 9 (old) from PCR: 5µL + 1µL loading dye

Deposit plan:

Ladder (5µL)
pCold 1 (12µL)
pCold 2 (12µL)
pCold 3 (12µL)
pHeat 1 (12µL)
pHeat 2 (12µL)
pHeat 3 (12µL)
PCR 9 (old) (6µL)
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

pCOLD (1033bp): 1200 bp

pHEAT (1694bp): 1 band at 1700bp (light) and 1 at 400bp

10/10 - 10/11 - Characterisation Protocole 0': Measure of the kinetic apparition of AmilCP

Objective: To measure the kinetic of AmilCP.

Protocol and Results:

We decided to carry on a kinetic of protein expression during 24hours to measure the influence of the DSBox on AmilCP expression by comparing the seq1 and 2, but the manipulation was unsuccessful and protein extraction was very compromised, hence we decided to proceed the Cold-shock characterisation with colorimetric measures only, waiting for our results through the iGEM Bettencourt collaboration and Nicolas Cornille's help.

10/11 - PCR on sequence 3 and Protegrin-1

Objective: Amplification of Protegrin-1 (BBa_K628006) (418 bp) and sequence 3 (BBa_K228007) (1091 bp).

Part 1: PCR

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Transformed colonies (on solid LB medium)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
Micropipettes and tips

Protocol:

We divided our 50µL mix into two 25ul mixes. The PCR is then performed with only 18 cycles to reduce the risk of errors (Taq polymerase not very accurate). Finally the two PCR products are mixed together before the clean up.

Pre-dilution Primers 100µM: 1,2 µL primer + 10,8 µL dH2O → 10µM final, volume total final

Pre-dilution Taq Polymerase 5000U/mL: 1,2µL Taq + 10,8µL dH2O → 500U/mL final, volume total final

For a 50µL PCR mix volume:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	5 µL	1X(1/10)
10mM dNTPs	1 µL	200µM (1/50)
10µM Prefix Primer	5 µL	1µM
10µM Suffix Primer	5 µL	1µM
Taq DNA Polymerase 500U/mL	5 µL	2,5 units/50 µL PCR
DNA template (10ng/µL)	2 µL
Nuclease-free water	27 µL	qsp 50ul

PCR program on the thermocycler:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
18	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	seq 3: 1 min protegrin-1: 30 sec(30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Gel migration

We made an agarose gel according to the protocol (please refer to our "Protocols" section). We are going to use 2/4µl of the PCR products to quantify the sequences concentrations efficiently.

Mixes for deposit: 7µL PCR product + 1,4µL 6X loading dye → 8,4µL total

2µl DNA: 2,4µl of the mix

4µl DNA: 4,8µl of the mix

Deposit plan:

Ladder
protegrin-1 (2µL)
protegrin-1 (4µL)
sequence 3 (2µL)
sequence 3 (4µL)
Ladder

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

We obtained two bands for the protegrin-1 at the expected size (between 400 and 600 bp), but no bands for the sequence 3, we suppose PCR has failed for this one.

10/11 - Digestion protegrin-1 + Ligation

Objective: To digest the sequence protegrin-1, and then ligate it into pSB1C3 backbone.

Part 1: Digestion

Material:

Sequence protegrin-1
Plasmid pSB1C3-mRFP (BBa_J04450 from IGEM Kit)
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We prepared the mixes according to the following instructions:

Reagents	Quantity	Final concentration
EcoRI-HF (20000U/mL)	1 µL	80U/µL
PstI (20000U/mL)	1 µL	80U/µL
Purified sequence	30 µL	10ng/µL
10X NEB buffer 2.1	5 µL	1X
Ultrapure water	13 µL

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

Part 2: Ligation

Material:

Digested insert (protegrin-1 and backbone pSB1C3)
Digested backbone (pSB1C3)
10X T4 DNA Ligase Buffer (NEB)
T4 DNA ligase (400000U/mL)
Ultrapure water
Eppendorf tubes
Micropipettes and tips

Protocol:

We prepared the mix for the ligation according to the following instructions (ratio 1:5):

insert (protegrin)	15,9µL
backbone pSB1C3	pSB1C3-mRFP (double digested, 25ng) 1,3µL
10X T4 DNA Ligase Buffer	2µL
T4 DNA Ligase	1µL
dH2O	6,1µL
Total volume	20µL

The sample was incubated overnight in the fridge.

10/12 - Transformation Protegrin + produits minipreps 1, 2, 7, 8, 9 + GFP constitutive (iGEM distribution kit)

Objective:

To amplify the ligation product of protegrin intro pSB1C3-mRFP, and select the right colonies to do minipreps and send to GATC
To cultivate the miniprep products of plasmids 1, 2, 7, 8 & 9 to assess prepare fresh cultures
To amplify the constitutive GFP plasmid to serve as a control for the pCOLD japanese test (plate 4 well 18A)

Material:

Ligated plasmids
Competent cells (DH5-alpha)
LB medium liquid and solid
SOC medium
Chloramphenicol
Ice
Eppendorf tubes
Micropipettes + tips
Waterbath
Petri dishes
Glass beads
Incubator
Centrifuge

Protocol:

Clean your working area by wiping down with 70% ethanol.

Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

Tubes	DNA	Plate	Competent cells	SOC medium
Protegrin	20 µL	LB + Chloramphenicol	50 µL	930 µL
1	10 µL	LB + Chloramphenicol	50 µL	940 µL
2	10 µL	LB + Chloramphenicol	50 µL	940 µL
7	10 µL	LB + Ampicillin	50 µL	940 µL
8	10 µL	LB + Chloramphenicol	50 µL	940 µL
9	10 µL	LB + Chloramphenicol	50 µL	940 µL
GFP	3 µL	LB + Kanamycin	50 µL	947 µL
Ligase +	20 µL	LB + Chloramphenicol	50 µL	930 µL
Ligase -	20 µL	LB + Chloramphenicol	50 µL	930 µL

Pipet DNA in each tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 940 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour 30min shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and 850µL of supernatant was discarded.

Bacteria were resuspended into the remaining culture media and bacteria were spread in the corresponding petri dishes.

Important notes:

The petri dishes we wanted to use were dried and we noticed that during the SOC incubation. We hence made new dishes quickly but the cultures had to stay in the SOC about 2h longer than usual. This is supposingly not impacting the cultures.
We however incubated 100µl of each culture (without centrifugation) on the dried plate to ensure security.

Results:

Interpretation:

Results of the transformation with GFP constitutive under UV light after 2 days of incubation at 37°C, in comparison with a transformation with sequence 7 (negative control).

The results are not significant.

As for the dishes made with long incubated bacteria in SOC medium, the dishes were crowded and uniform.

10/12 - Cold shock characterisation

Objective: 100µl of each transformed culture has been incubated in corresponding LB+ antibiotics medium. The objective is to measure the growth of the cold shock through optical density (OD) at 600 nm and then when it reaches 0.5 at 600 nm, extract the proteins and measure OD at 588 nm.

Protocol:

For all measures, 1,5 ml of liquid culture were taken from cultivated medium.

Results:

OD600 8h30 PM:
OD 600 Sequence 7: 0.013 (We added 1ml of fresh seq 7 that was incubating since the 10/11 to "boost" the growth and avoid any time shift. The bacteria came from the same original glycerol stock. The addition was not sufficient and we added 1 more mL that we however pelleted into 200µl before).

OD 600 9h40 PM:
OD 600 Sequence 7: 0,139 -The boost seems to have worked but not enough.
OD 600 Sequence pCold: 0,522 → 9h53 PM: one cuve sent to be observed under fluorescence, then we divided the volume in two separate erlenmeyer with one at 15°C and the other at 37°C.

OD600 11h10 PM:
OD 600 Sequence 7: 0.285 (0,325 after adding 1,0ml of sequence 7 pelleted into 200µl from 11/10 coming from the same glycerol stock)

Sequences	OD 600 17H20	OD600 19H	OD 600 20H30	OD600 21H40	OD600 23H10
Sequence 1	0.004	0.055	0.166	0.307	0,486
Sequence 2	0.008	0.041	0.155	0.298	0,479
Sequence 7	0.007	0.013	0.013	0.139	0,285
pCold	0.023	0.167	0.370	0.522

After the OD600 measurements, 40mL was left in each erlenmeyers, we splitted the cultures in two. For each cultures we obtained 20mL liquid medium with our bacteria, 20mL was put at 15°C and the other was kept at 37°C.

Incubation sequences 1 and 2 at 15°C at 23H45 (divided in 2 with one half at 37°C and another at 15°C).

OD600 and incubation at 00h15 AM:
Sequence 7: 0,464 ==> divided in 2 with one half at 37°C and another at 15°C .

Time of incubation split:

pCOLD: 9h53 PM
Sequence 1: 11h45 PM
Sequence 2: 11h45 PM
Sequence 7: 00h15 AM

10/12 - Minipreps sequence pCold and pHeat

Objective: To make stocks

Material:

Monarch® Plasmid Miniprep Kit (NEB)
Micropipettes + tips
Eppendorf tubes
Centrifuge
Ice

Protocol:

The same protocol as usual was used.

NB: Pay attention to the elution volume. We eluted in 60µL nuclease-free water and not 30µL.

10/13 - Liquid culture of transformed bacteria

Protocol:

Liquid cultures of the transformation obtained from the first batch of plate (on semi-dried petri dishes) were made with 10ml of liquid LB supplemented with appropriate antibiotics:

The colonies were selected based on their colors. The GFP transformed colonies emitted no color but were transformed with pure plasmid from the iGEM plates so we were confident no other colonies than the transformed ones would be on the plate.

Seq 1/2/8/9 - Chloramphenicol

Seq 7 - Ampicillin

GFP Constitutive - Kanamycin

Results:

The ligase control shown appropriate results.

The culture liquid of the Protegrin transformed bacteria was not made since we had to wait for the results of a PCR Colony to proceed.

UPDATE:

Half the cultures had grown after 24h, we assumed the RPM were not optimal and the culture might have been badly poured into the medium at first, another batch will be done on the 10/14.

10/13 - PCR colony on Protegrin

Objective: Select the colonies transformed with the plasmid pSB1C3-protegrin-1 to further purify it and send it to GATC.

Part 1: PCR colony

Material:

10X Standard taq reaction Buffer (NEB)
10µM dNTPS (NEB)
Forward primer 100µM (synthesized by IDT)
Reverse primer 100µM (synthesized by IDT)
Taq DNA polymerase (5000U/mL)
DNAse/ RNAse free water
PCR tubes
Thermocycler
micropipettes and tips

Protocol:

We picked:

for protegrin-1: 3 colonies (white) (418bp)

for the control: 1 pSB1C3-mRFP (1069bp)

Pre-dilutions (1/10):

Primers: 1µL + 9µL dH2O → 10µM final

Taq Polymerase 5000U/mL: 1µL + 9µL dH2O → 500U/mL final

Each chosen colony has been annotated on the petri dishes so we will be able to use them again for liquid culture later. They are now stocked in the fridge, protected by parafilm.

Cell membrane destruction:

In each PCR tube: put 8µL of ddH20. Pick a colony (with a toothpick or a micropipette tip), add a stab of it into the PCR tube.

Incubate the tubes at 95°C for 5min.

Tube mRFP: put 2µL DNA template

Prepare the PCR mix:

Component	Volume	Final concentration
10X Standard Taq Reaction Buffer	8µL	1X
10mM dNTPs	1,6µL	200µM (1/50)
10µM Preffix Primer	8µL	1µM
10µM Suffix Primer	8µL	1µM
Taq DNA Polymerase	8µL	2,5 units/50µL PCR
Nuclease-free water	46,4µL	qsp 80µL

Put 17µL of mix in each colony tube (25-8=17)

Put 23µL in the mRFP tube (25-2=23)

PCR thermocycler program:

PCR Cycle
Repetition	Step	Time	Temperature
0	Initial denaturation	30 sec	95°C
30	Denaturation	30 sec	95°C
	Annealing	1 min	60°C
	DNA polymerization	protegrin-1: 30 sec mRFP: 1 min (30 sec/kb)	68°C
0	DNA polymerization	5 min	68°C
	FINISH	∞	4°C

Part 2: Gel migration

We made an agarose gel according to the protocol (please refer to our "Protocols" section).

Preparation of the samples for the migration: 10µL sample + 2µL 6X loading buffer

Deposit plan:

Ladder (5µL)
Protegrin-1 1
Protegrin-1 2
Protegrin-1 3
Ladder (5µL)

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

The colony 2 seems better (the band is more visible).

10/13 - Cold shock characterisation

Objective: To measure the growth of the cold shock through Optical Density (OD) at 600 nm and then when it reaches 0.5 at 600 nm, extract the proteins and measure OD at 588 nm.

Protocol:

For all measures, 1,5 ml of liquid culture were taken from cultivated medium.

Beginning of extraction: 5pm → Total of approximately 18h of incubation at 15°C and 37°C for all cultures.

Visual results: we pelleted 1.5ml of each culture

Wild-type DH5alpha (ancient culture in liquid LB at 37°C)

Results:

Interpretation:

A: Wild-type DH5 alpha (ancient culture in liquid LB at 37°C)

B: Sequence 7 at 15°C (Left) and 37°C (Right): We can clearly see AmilCP expression at 15°C, so this is good for us ! Note the greyish aspect for the 37°C pellet, implying a potential low expression of the protein (but still very low as compared to the 15°C pellet).

C: Another sneak peak at our sequence 7 expected results.

D: Seq 2 at 15°C (left) and at 37°C (right): Here the difference is not exceptional but the right pellet coming from the culture at 37°C is greyer than the left one, showing "better" AmilCP expression and coherent results.

The poor expression is supposedly due to the long time storage in LB of our Seq 2 transformed colonies, we hence will re-do the protocol with fresh transformed bacteria and analyse.

E: Seq 1 at 15°C (left) and 37°C (right): exact same results as for the sequence 2, we still need confirmation with fresh bacteria.

F: pCOLD: The left pellet seems a little fluorescent as compared to the right one. Efficiency of the response will be further seen on the 10/14 and with the Bettencourt collaboration.

Protein extraction results:

Seq 1 - 15°C: DO = 0.012
Seq 2 - 15°C: DO = 0.025
Seq 7 - 15°C: DO = 0.007
Seq 1 - 37°C: DO = 0.017
Seq 2 - 37°C: DO = 0.011
Seq 7 - 37°C: DO = 0.014

10/14 - Cold shock characterisation

Objective:We end this first characterisation by a final visual measure on our culture. We decided to skip the protein extraction protocol since it provided very few results on the 10/13 and was not reliable.

Protocol:

Hour of end: 5h11 pm

Total: 37-44h

Results:

OD600 Seq 1 37°C: 0,690
OD600 Seq 1 15°C: 0,688
OD600 Seq 2 37°C: 0,685
OD600 Seq 2 15°C: 0,674
OD600 Seq 7 37°C: 0,715
OD600 Seq 7 15°C: 0,615
OD600 Seq pCOLD 37°C: 0,698
OD600 Seq pCOLD 15°C: 0,674

Interpretation:

OD600 are almost equal between all samples, showing that the maximal growth plateau has been reached. These similar values allow a relevant comparison of cells coloration, as pellets contain approximately the same number of bacteria.

Once again, this experiment proves the efficiency of the cold-shock plasmid (pSB1C3-seq7): no coloration was observed at 37°C after 37h, whereas a strong blue coloration appeared during the same time at 15°C.

In comparison, the sequences 1 and 2 coding for constitutive amilCP expression remained white at 15°C and gave a slight blue coloration at 37°C. At 15°C, the absence of coloration can be explained by the cold temperature slowing down the metabolic process. At 37°C, we should observe a stronger coloration. However, amilCP takes approximately 30 hours to be fully functional. After 40h, it is possible that the amilCP expression was just at its beginning. We should therefore reproduce the experiment after a longer incubation. Indeed, some liquid culture put at 37°C for several days displayed a strong blue coloration.

Week 9

10/16 - Miniprep of sequences 1/2/7/8/9/Protegrin/Constitutive GFP of 14/10 cultures

Objective: To stock our plasmids to send them to the iGEM. We are going to migrate a sample of each miniprep to make sure they are present. Some of the protegrin miniprep will also be sent to GATC to be sequenced.

Material and Protocol:

The same protocol as usual was used for the miniprep of each plasmid.

10/16 - Cold shock characterisation Duplicat 1 - Day 1

Objective: To obtain the same results as last week a second time in order to confirm those results.

Protocol:

This time no pCOLD culture was made. We assessed it would be more efficient to quantify pCOLD effect with the use of microplate reader for the GFP.

We prepared then:

One culture of 1ml of seq 1 from the 10/14 liquid culture in 50ml LB medium + chloramphenicol
One culture of 100µl of seq 2 from a glycerol stock in 50ml LB medium + chloramphenicol. We took the glycerol stock because of unwished events with the 10/14 culture. We later took the 10/13 culture to enhance the growth of the former, because it was very slow. Both cultures came from the same origin and were transformed by the same plasmid.
One culture of 1ml of seq 7 from the 10/14 liquid culture in 50ml LB medium + ampicillin

OD600 at t=0h
Seq1: 0,062
Seq2: 0,010
Seq7: 0,038

OD600 at t=1h30
Seq1: 0,057
Seq2: 0,010, we added 1ml of 13/10 and reached 0,031
Seq7: 0,092

OD600 at t=3h
Seq1: 0,077
Seq2: 0,029
Seq7: 0,322

From this we assessed we would measure the next OD600 of seq 7 in one hour and leave the other incubate more.

OD600 at t=4h
Seq7: 0, 463

Alright ! We splitted the culture in two erlenmeyer of 20ml, one was put at 15°C and the other at 37°C for 24h

Time of incubation: 16h20

OD600 at t=5h
Seq1: 0,195
Seq2: 0,103

OD600 at t=6h30
Seq1: 0,357
Seq2: 0,257

From this we assessed we would measure the next OD600 of seq 1 in one hour and leave the seq2 incubate more

OD600 at t=7h30
Seq1: 0,473

We splitted the culture in two and incubated one erlenmeyer at 37°C and the other at 15°C

Time of incubation: 19h10

OD600 at t=8h15
Seq2: 0.488

Time of incubation: 20h05

10/16 - Competent cells by CaCl2 - Day 1

Objective: To make competent cells.

Protocol:

We incubated wild-type DH5 alpha from the IGEM 2016 stock into 15 ml of liquid LB medium without antibiotics.

10/17 - Migration of minipreps from 10/16

Objective:We decided to migrate 5µl of each miniprep from the 10/16 to ensure everything was here before sending anything to GATC (protegrin) or the IGEM registry. This migration will also permit us to quantify the pSB1A3-Seq7 and pSB1K3-GFP before following digestions and ligations into pSB1C3. We also migrated a pSB1C3-mRFP control.

Material:

Agarose
Distilled water
TAE buffer 50X
Gel cast and combs
Electrophoresis cuve
Microwave
Micropipettes and tips
2-Log DNA Ladder (0.1-10.0 kb) (NEB)
Gel loading dye purple 6X (NEB)

Protocol:

Sample preparation: 5µl of each sample + 1µl of DNA 6X Loading dye.

We deposited 5µl of each sample in the gel and 5µl of Ladder.

We made an agarose gel according to the protocol (please refer to our "Protocols" section).

Deposit plan:

Ladder
pSB1C3-Seq1
pSB1C3-Seq2
pSB1A3-Seq7
pSB1C3-Seq8
pSB1C3-Seq9
pSB1K3-GFP
pSB1C3-Protegrin
pSB1C3-mRFP
Ladder

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Usual average size:

pSB1C3-Seq1: 3000bp
pSB1C3-Seq2: 3000bp
pSB1A3-Seq7: 3000bp
pSB1C3-Seq8: 3000bp
pSB1C3-Seq9: 4000bp
pSB1K3-GFP: 2989 + 919 = about 3800bp
pSB1C3-Protegrin: 2500bp
pSB1C3-mRFP: 300bp

Results:

Interpretation:

If we refer to the control, the seq 8/9/7/Protegrin in their respective plasmids all migrated as predicted. However Seq 1 and 2 seems to be very high (4000bp). The GFP seems a little low too. This migration pattern could be explained by the fact that plasmid migrate sometimes not as planned and that there are different plasmid forms in each well. Maybe the heavier plasmid form (Nicked) predominates totally for seq 1 and 2 and the lighter (linear) predominates for the GFP.

There are important smears for sequences 1/2/7/9.

Importantly enough if we look at the ladder on the right, the size seems more adequate and the unexpected bands could be entirely explained by plasmid forms.

10/17 - Cold shock characterisation Duplicat 1 Day 2

Protocol:

At 10 AM, we took the seq 7 at 15°C and 37°C and pelleted 1,5ml after measuring the OD600:

OD600 seq 7 at t = 18h:
37°C: 0,685
15°C: 0,576

We then we took the seq1 and seq2 out at both 37 and 15°C, 0D600 of 1,5ml of each was measured and then was pelleted:

OD600 seq 1 at t = 18h:
37°C: 0,707
15°C: 0,587

OD600 seq 2 at t = 18h:
37°C: 0,694
15°C: 0,618

Results:

Interpretation:

For sequence 7, again we can clearly see AmilCP expressed for the incubated transformed bacteria at 15°C (blue). We can distinguish a greyish pellet at 37°C, indicating AmilCP expression but in low quantity.

10/17 - Competent cells by CaCl2 - Day 2

Day 2

1. Inoculate 100 mL of LB with 1 mL of the starter culture in a 500 mL Erlenmeyer

2. Place some 50 mL falcon tubes and 1.5 mL eppendorf tubes on ice and put them in the fridge at 4°C. Turn on the centrifuge and cool the rotor to 4°C.

3. Incubate at 37°C with shaking until the cells reach early exponential phase, it takes 1- 3h until the culture becomes slightly turbid (OD600~0.4). Do not let the culture grow more than OD600 0.4.

We incubated 1 ml of wild-type DH5 alpha from yesterday's culture into 100 ml of liquid LB medium without antibiotics and we measure the OD600 every 1h30.

Time	OD600
0	0,036
1h30	0,073
3h	0,457

4. Aliquot into 50 mL pre chilled tubes and centrifuge 15 min at 4000 rpm in the big centrifuge at 4°C. Decelerate without brake. Discard supernatant.

KEEP THE CELLS IN ICE ALL TIMES AND DO NOT VORTEX.

5. Resuspend GENTLY in 25 mL cold MgCl2 100 mM. Centrifuge cells 15 min at 4000 rpm in the big centrifuge at 4°C. Decelerate without brake. Discard supernatant.

6. Resuspend GENTLY each pellet in 10 mL ice-cold CaCl2 100 mM solution. Keep resuspended cells on ice and place the box in the fridge overnight at 4.

10/17 - Heat response characterisation - Pre culture

Protocol:

Pre-culture of seq 8 and 9 were made in 15ml falcons (3ml of LB + chloramphenicol AMl of culture from 10/14). Incubation was made at 37°C O/N. This is to prevent any long wait caused by any unfreezing of glycerol stocks that could lead to long incubation time to reach OD600 = 0.5.

10/18 - Digestion recombinant plasmids for gel migration

Objective: To digest our recombinant plasmids in order to analyze them on an electrophoresis gel.

Part 1: Digestion

Material:

DNA to digest
EcoRI-HF (NEB: 20000U/mL)
PstI (NEB: 20000U/mL)
10X NEB Buffer 2.1
Ultrapure water
1,5mL Eppendorf tubes
Micropipettes + tips
Waterbath
Heat Block

Protocol:

We perform this digestion on plasmids containing: Seq 1, 2, 7, 8, 9, protegrin and GFP

Preparation of the mix:

5µL of miniprep product
1µL EcoRI HF (20000U/mL)
2,5µL 2.1 NEB Buffer 10X
16,5µL H2O

Digestion was carried out 2 hours at 37°C

Heat inactivation was proceeded at 80°C for 20 minutes.

Part 2: Gel migration

We made an agarose gel according to the protocol (please refer to our "Protocols" section)

Preparation of the samples for deposit: 5µL of digestion product + 1µL 6X loading dye

Deposit plan:

Ladder
pSB1C3 seq 1
pSB1C3 seq 2
pSB1A3 seq 7
pSB1C3 seq 8
pSB1C3 seq 9
pSB1K3 GFP
pSB1C3 protegrin
Ladder

Gel migration was performed during 1h30 at 110V.

The gel was incubated in a GelRed bath during 1h30.

Results:

Interpretation:

After the migration, as compared to the previous gel, all the bands seem to be at the right height. The GFP is higher because of the weight of pSB1K3 (2204bp). The protegrin is located correctly (around 2500bp). The only strange result comes from the seq 2 which presents two bands. That does not make sense and an explanation could be that our plasmid is mutated and that this mutation induced the formation of an EcoRI site, leading to the opening of the plasmid. We are going to use another miniprep of seq 2 that dates from the 08/30 to maybe try to get the unmutated sequence and pursue our experiments.

10/18 - Cold shock characterisation Duplicat 1 - Day 3

At 10am, we took the seq 7 at 15°C and 37°C and pelleted 1,5ml after measuring the OD600:

OD600 seq 7 at t= 42h:

37°C: 0,685

15°C: 0,656

We can clearly see AmilCP expressed for the incubated seq7-transformed bacteria at 15°C (blue). Even after 42h of incubation at 37°C, seq7-transformed bacteria do not express AmilCP.

We then we took the seq 1 and seq2 out at both 37 and 15°C, 0D600 of 1,5ml of each was measured and then was pelleted:

OD600 seq 1 at t = 42h:
37°C: 0,707
15°C: 0,587

OD600 seq 2 at t = 42h:
37°C: 0,694
15°C: 0,618

10/18 - Competent cells by CaCl2 - Day 3 and transformation test with NEB kit

Day 3

1. Make sure the centrifuge is cold and harvest the cells by centrifuging 15 min @4000 rpm in the big centrifuge at 4°C. Decelerate without brake. Discard supernatant and GENTLY resuspend each pellet in 10 mL ice-cold 100 mM CaCl2 + 15% glycerol solution.

2. Centrifuge cells 15 min at 2500 rpm 4°C. Discard supernatant and GENTLY resuspend each pellet in 2 mL ice-cold 100 mM CaCl2 + 15% glycerol solution.

3. Make aliquots with 100 uL each. Freeze immediately at -80°C.

Transformation with cell test kit from NEB:

Pipet 1µl of DNA from the 100µg/µl tube of the NEB Kit in a tube and then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

We also did a blank with 50µl of competent cells and 1µl of water.

Incubate on ice for 30 minutes. Put it in the fridge, the temperatures in the room are too high.

Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

Heat-shock the cells by placing into the waterbath for 30 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. IN THE FRIDGE AS WELL

Add qs 940 µL of SOC media WITHOUT CHLORAMPHENICOL per tube, and incubate at 37°C for 1 hour 30min shaking at 200-300rpm.

Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture. Put the agar plates at 37°C.

Tubes were centrifuged at 3500rpm for 4minutes and 850µL of supernatant was discarded. Bacteria were resuspended into the remaining culture media and bacteria were spread in the corresponding petri dishes.

One petri dish with chloramphenicol and one petri dish without antibiotics was hence incubated.

Important notes:

The petri dishes we wanted to use were dried and we noticed that during the SOC incubation. We hence made new dishes quickly but the cultures had to stay in the SOC about 2h longer than usual. This is supposingly not impacting the cultures.
We however incubated 100µl of each culture (without centrifugation) on the dried plate to ensure security.

10/18 - Heat shock characterisation - Day 1

Objective: To measure mRFP expression by seq9-transformed bacteria depending on the temperature at 27°C and 37°C.

Protocol:

We prepared:

One culture of 2ml of seq 8 from the 10/17 liquid pre-culture in 100ml LB medium + chloramphenicol
One culture of 2ml of seq 9 from the 10/17 liquid pre-culture in 100ml LB medium + chloramphenicol.
One culture of 2ml of WT DH5-alpha from the 10/14 liquid culture in 50ml LB medium + ampicilin

We start the incubation at T=0 (14h15) and every 1h30 we measured the OD600 until 0D600=0,4.

OD600 at t=0h
Seq8: 0,058
Seq9: 0,040
WT: 0,062

OD600 at t=1h30
Seq8: 0,075
Seq9: 0,047

WT: 0,113

OD600 at t=3h
Seq8: 0,190
Seq9: 0,054
WT: 0,344

We stopped the experiment at this point because of several technical problems. (Multitron doesn't accept erlens > 500ml and seq 9 wasn't growing correctly). We decided to launch a second time the experiment the 10/19 and we let the bacteria O/N at 37°C 250RPM.

10/19 - Bacterial Transformation with pSB1C3-seq2

We start a transformation using competent cells from 10/18 and miniprep of seq2-PSB1C3 from 30/08.

Transformation of competent cells with pSB1C3-Seq2:

Pipet 5µl of DNA from the then 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.