Team:Missouri Rolla/Notebook

Missouri_Rolla


30 October 2017

Ryan Baumann

Start: 5:00PM


Cloning efficiency testing of new Universal Acceptor Plasmid


Purpose: To test the cloning efficiency of the Universal Acceptor Plasmid after deletion of the unexpected 10 bp region via site directed mutagenesis PCR

Protocol:


Golden gate assemblies were performed to insert TNTR3 gblock into our universal acceptor plasmid. Various molar ratios of TNTR3: Universal acceptor plasmid were tested. Molar ratios of 1:2, 1:1, 2:1 (recommended ratio), 4:1, and 8:1 were tested for the new plasmid. A 2:1 ratio assembly was performed using our original UAP. A negative control assembly was performed with no inserts added. Golden gate assemblies contained the following: 1:2 Ratio
ComponentVolume
TNTR3 (17.64 ng)
New UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
1:1 ratio
ComponentVolume
TNTR3 (35.28 ng)
New UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
2:1 Ratio
ComponentVolume
TNTR3 (70.56 ng)
New UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
4:1 Ratio
ComponentVolume
TNTR3 (141.12 ng)
New UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
8:1 Ratio
ComponentVolume
TNTR3 (282.24 ng)
New UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
2:1 Ratio with original UAP
ComponentVolume
TNTR3 (70.56 ng)
Original UAP (100ng)1 μL
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
Negative Control
ComponentVolume
TNTR3None
UAP None
10X Ligase Buffer2 μL
Ligase1 μL
BsmBI1 μL
MilliQ H2Oup to 20μL
All samples were run with the following thermocycler conditions: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C Following assembly, 1 uL of each sample was transformed on to Chloramphenicol Blue-White Screening plates following Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. Dillutions of 20% and 5% were plated and spread using sterile glass beads. A positive control transformation was performed using 1 uL of original UAP. Colonies were incubated at 37C for 30 hours. Blue and white colonies were counted on all plates.

Stop: 1:45AM


Results:


See project page

Next:


Boston!

25 October 2017

Ryan Baumann

Start: 3


Blunt end Ligation of 10/22/17 PCR1 and transformation


Purpose: Ligation of UAP back to itself after removal of 10bp sequence.

Protocol:


Blunt end ligation was performed uisng NEB T4 DNA ligase protocol. 2 uL of 10/22/17 OJM PCR 1 was ligated in 20 uL reaction. Reaction was conducted at room temperature for 2 hours. After Ligation, 2 uL of 10/25/17 L1 was transformed using Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. 50% and 5% dilutions were plated onto Chloramphenicol plate.

Notes:


Check colony growth

Stop: 5:00


Products:


LabelSource Description
10/25/17 L110/22/17 PCR1UAP Ligated back to itself following removal of 10bp fragment

Next:



23 October 2017

Ryan Baumann

Start: 8:45PM


Colony PCR's of 10/18/2017 Transformations of PhoB and TNTr3, gels of Golden Gate Round two PCR's and 10/23/17 PCR 1 and 2


Purpose: To check assembly of golden gate round two assemblies and test OJM1 and OJM2 primers.

Protocol:


18 Colony PCRs were conducted using Taq 2x Master Mix protocol. 9 Colonies from PhoB and TNTR3 transformation plates were used as template. PCR 1-9 - TNTR3 PCR 10-18 PhoR Three 1% agarose gels were prepared with 2 uL of ethidium bromide Gel 1:
Lane(s)Component
1 2 Log purple DNA ladder
2TNTR3 PCR1
32 Log purple DNA ladder
4-10TNTR3 PCR 2-3 and 5-9
112 Log purple DNA ladder
12-20PhoR PCR 1-9
Gel 2:
Lane(s)Component
1 2 Log purple DNA ladder
2-810/23/17 cPCR's 1-7 (Q92X31)
132 Log purple DNA ladder
14-2010/23/17 cPCR's 8-14 (PhoB)
2310/23/17 OJM PCR1
Gel 3:
Lane(s)Component
12 Log Purple DNA Ladder
2-810/23/17 cPCR 15-21 (O22527)
112 Log Purple DNA Ladder
12-1810/23/17 cPCR 22-28 (Q8LDU4)

Notes:



Stop: 10:30


Next:



23 October 2017

Jeremy Mesa

Start: 5:00 pm



Purpose: To perform a PCR with mutagenesis primers in order to delete a short base pair sequence from plasmid.

Protocol:


Contents of PCR tubes:
Q5oJM1oJM2UAPMilli-Q
PCR 112.5 μL1.25 μL1.25 μL1 μL4 μL
PCR 2 12.5 μL1.25 μL1.25 μL0 μL5 μL
PCR 2 was a negative control. Thermal Cycler Program:
Temp (°C)TimeCycles
Step 19830 sec0
Step 2985 sec24
Step 36520 sec24
Step 47275 sec24
Step 5722 min0
Step 64Infinite

Stop: 5:45


Next:



23 October 2017

Ryan Baumann, Jessica Brooks, Tiffany Kuhnert, Lucas Dyer

Start: 4:15pm


Colony PCR of 10/23/2017 Q92x31 5%, Q22527 5% , PhoB 50% , and Q8LD44 5% , golden gate round 2 transformation plates


Purpose: To confirm assembly of title parts with their promoters and terminators

Protocol:


28 Colony PCRs were conducted using Taq 2x Master Mix protocol. 7 colonies from each plate were run along with a RFP Construct postitive control and milliQ water negative control. 1-7 Q92X31 8-14 PhoB 15-21 O22527 22-28 Q8LDU4 Reaction Set Up:
ComponentVolume (μ)
Taq 2x Master Mix7.5
VR0.3
VF20.3
MilliQ6.9
Colony1-34
Thermocycler Conditions:
Temp. (C)TimeRepeats
Step 1955 min0
Step 29530 sec35
Step 35130 sec35
Step 4683 min35
Step 5685 min0
Step 64

Stop:


Next:


Run Gels of all the colony PCRs

18 October 2017

Ryan Baumann

Start: 2:30PM


Plasmid mini preps of Q9LX31, Q8LDU4, and O22527 transformations. Golden gate assemblies of PhoB, PhoR, TNTR3, Q9LX31, Q8LDU4, and O22527 and transformation on to Kanamysin Blue white screening plates


Purpose: Purify degreening plasmids and assemble signal transduction pathway, periplasmic binding protein, and degreening parts into transcriptional units.

Protocol:


Minipreps were conducted using iGEM lab manual 2.5 kitless miniprep protocol. Samples were resuspended in 50 uL of TE buffer. DNA Concentration were calculated using Thermoscientic Nanodrop 1000. Nanodrop Results:
SampleDNA Conc. (ng/μL)260/280
10/17 MP3 (O22527)122.12.11
10/17 MP4 (O22527)364.91.86
10/17 MP5 (Q9LX31)326.91.88
10/17 MP6 (Q9LX31)280.21.91
10/18 MP1 (Q8LDU4)76.71.93
10/18 MP2 (Q8LDU4)59.31.90
10/18 MP3 (K1467101)47.41.93
10/18 MP4 (K1467101)100.31.93
Golden Gate Assemblies of PhoB (9/8/17 MP3), PhoR (9/13/17 MP1), TNTR3 (9/8/17 MP1), O22527 (10/17/17 MP4), Q9LX31 (10/17/17 MP5), Q8LDU4 (10/18/17 MP2) were performed following the NEB Golden Gate assembly kit protocol into golden braid alpha 1 acceptor plasmid (BBa_P10501). The plant pho promoter (9/8/17 MP2) was added to the degreening parts. BBa_K1467101 added to TNTR3, PhoR, and PhoB. BBa_K1618037 (9/13/17 MP3) was added to all parts. 2 uL of each golden gate assemblies were transformed using Intact Genomics ig 5-Alpha chemically competent cell protocol. 50% and 5% dilutions were plated onto Kanamysin Blue white screening plates.

Stop: 8:30PM


Results:


Check for colonies

Products:


Label SourceDescription
10/17 MP3O22527O22527 in the UAP
10/17 MP4O22527O22527 in the UAP
10/17 MP5Q9LX31Q9LX31 in the UAP
10/17 MP6Q9LX31Q9LX31 in the UAP
10/18 MP1Q8LDU4Q8LDU4 in the UAP
10/18 MP2Q8LDU4Q8LDU4 in the UAP
10/18 MP3K1467101K1467101 in the UAP
10/18 MP4K1467101K1467101 in the UAP
10/18 GG19/8/17 MP3PhoB with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG29/13/17 MP1PhoR with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG39/8/17 MP1TNTR3 with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG410/17 MP4O22527 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG510/17 MP5Q9LX31 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG610/18/17 MP2 Q8LDU4 with Plant Pho Promoter and BBa_K1618037 Terminator

Next:



17 Oct. 2017

Jeremy Mesa

Start: 2:30



Purpose: To run PCR with mutagenesis primers and a gel following to visualize the results of the reaction.

Protocol:


PCR: Contents of the tube, 20 μL total
TubeQ5oJM1oJM2UAPMilli-Q
PCR 110 μL1 μL1 μL1 μL7 μL
Thermal Cycler Program:
Temp. (°C)TimeRepeats
Step 19830 sec0
Step 29810 sec30
Step 36520 sec30
Step 4722 min30
Step 5722 min0
Step 64Infinite0
1% Agarose gel was prepared with 3 μL Ethidium Bromide.
Lane 1Ladder20 μL
Lane 4PCR 115 μL

Notes:


Program name on thermal cycler is saved as PCR10-6.

Stop: 4:00


Results:


PCR lane of gel was found to have 4 bands at about 2500bp, 1500bp, 600bp, and less than 100bp. The lane was streaked behind the largest base pair band.

Next:



10 15 2017

Lynell Cunningham, Luke Dyer, Tiffany Kuhnert

Start: 3:00


Colony PCR and Gel of 9/30/17 Golden gate assemblies of degreening parts


Purpose: To confirm assembly of Q9LX31, Q8LDU4, and O22527 into the universal acceptor plasmid.

Protocol:


17 Colony Pcr's were conducted using Taq 2x Master mix protocol. 5 Colonies from each 9/30/17 golden gate reaction were run along with a RFP Construct postitive control and milliQ water negative control. Reaction setup:
ComponentVolume
Taq 2X Master Mix10 μL
VR.4 uL
VF2.4 uL
MilliQ9.2 uL
Colony 1-15
Thermocycler Conditions: 95C for 5 minutes, 35 Cycles of: (95C for 20 seconds, 66C for 30 sec, 68C for 1min), 68C for 5 minutes, and 4C hold All samples were run on a 1% agarose gel with the following setup: Lane 1 and 11: 2 log purple DNA ladder Lane 2-6 are Colonies 1-5 from Q8LDU4 Lanes 7,8,9,10,and 12 are Colonies 6-10 from O22527 Lanes 13-17 are Colonies 11-15 for Q9LX31 Lane 18 is a water negative control Lane 19 is the RFP Construct positive control After imaging, one colony from each golden gate transformation was grown in broth culture containing Chloramphenicol

Notes:



Stop: 10:00pm


Next:


Grow successfully assembled cultures, miniprep, and round two assemblies

10 October 2017

Ryan Baumann

Start: 4:30PM


Gel of 9 October 2017 Golden Gate and Colony PCR's


Purpose: To confirm proper assembly of previous golden gate assemblies

Protocol:


A 1% agarose gel with 1.5 μL Ethidium Bromide and 2 Log Purple DNA Ladder.10 μL of samples were loaded into wells.
WellSample
12 Log purple DNA Ladder
21
32
43
54
65
76
87
98
109
112 Log Purple DNA Ladder
1210
1311
1412
1513
1614
1715
1816
1917
2018
2110/9/17 A1
2210/9/17 A2
2310/9/7 A3

Notes:



Stop: 6:00:00PM


Results:


Analyze Gel Results

Next:



30 September 2017

Kent Gorday, Lynell Cunningham, Lucas Dyer, Jeremy Mesa

Start: 16:35


Diagnostic PCRs of round 2 assemblies and assembly of degreening gblocks into UAP


Purpose: To check addition of constitutive promoter and terminator to coding sequences, and assemble degreening coding sequences into Universal Acceptor Plasmid for submission.

Protocol:


Six PCRs were performed from round 2 assemblies:
9/30 A1-6
10 μL2X Q5 Master Mix
7 μLMilliQ Water
1 μLTemplate Plasmid
1 μLVF2
1 μLVR
20 μLTotal Volume
Temperature (°C)Time (sec)
9830
98c8
65c22
72c93
72120
4

cycle X32

Three Golden Gate reactions into the Universal Acceptor Plasmid were performed:
9/30 GG1-3
4.9 μL100 ng/μL Q9LX31 gblock
2.5 μLUAP
2 μL10X T4 DNA Ligase Buffer
1 μLT4 DNA Ligase
1 μLBsmBI
8.6 μLMilliQ Water
15.1 μLTotal Volume
5.9 μL100 ng/μL O22527 gblock
2.5 μLUAP
2 μL10X T4 DNA Ligase Buffer
1 μLT4 DNA Ligase
1 μLBsmBI
7.6 μLMilliQ Water
14.1 μLTotal Volume
5.8 μL100 ng/μL Q8LDU4 gblock
2.5 μLUAP
2 μL10X T4 DNA Ligase Buffer
1 μLT4 DNA Ligase
1 μLBsmBI
7.7 μLMilliQ Water
14.2 μLTotal Volume
Temperature (°C)Time (sec)
3720
37c180
16c240
50300
80300
16300

cycle X32

A 1.2% agarose gel was run with PCR products:
Well No.
3Ladder
4A1
5Ladder
6A2; Bad Load
7A4
8A3; Bad Load
9A5
10A6

Stop: 20:40


Results:



Products:


LabelSourceDescription
9/30 A19/18 MP1 + pro/ter amplified
9/30 A29/18 MP2 + pro/ter amplified
9/30 A39/18 MP3 + pro/ter amplified
9/30 A49/18 MP4 + pro/ter amplified
9/30 A59/18 MP5 + pro/ter amplified
9/30 A69/18 MP6 + pro/ter amplified
9/30 GG1Q9LX31 gblockGUN4 CDS assembled into Universal Acceptor Plasmid
9/30 GG2O22527 gblockChlorophyllase-1 CDS assembled into Universal Acceptor Plasmid
9/30 GG3Q8LDU4 gblockRCCR CDS assembled into Universal Acceptor Plasmid

Next:


Colony PCR and patch to a new plate a few white colonies from each round 2 assembly transformation.

9-21-17

Ryan Baumann

Start: 1:30PM


PCR and Gel of 18 September 2017 minipreps (again)


Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:


Six 20 μL PCR's of 9/18/17 MP 1-6 were run using containing: Q5 HIgh Fidelity 2X Master Mix, 0.4 μL VR and VF2 (10 μM solutions), The following template concentrations, and MilliQ Water up to 20 μL.
TemplateVolume
9/18/17 MP11 μL
9/18/17 MP21 μL
9/18/17 MP31 μL
9/18/17 MP41 μL
9/18/17 MP54 μL
9/18/17 MP65 μL
A positive control was run using a 1μL of RFP construct in psb1C3. A negative control was run using MilliQ water. Samples were run in the thermocycler in the following conditions: 98C for 30 seconds 32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds 72C for 120 seconds 4C Hold All PCR's were run on a 1% agarose gel containing 1.5 μL of ethidium bromide
WellSample
12 Log Purple DNA Ladder
3PCR1(MP1)
4PCR2(MP2)
5PCR3(MP3)
6PCR4(MP4)
7PCR5(MP5)
8PCR6(MP6)
9Positive Control
10Negative Control

Notes:



Stop:


Results:


Continue to troubleshoot PCR conditions. No conclusive results from gel.

Next:


Redo PCR's again

21 September 2017

Ryan Baumann

Start: 2:00 PM


PCR and Gel of 18 September 2017 minipreps


Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:


Six 20 μL PCR's of 9/18/17 MP 1-6 were run using containing: 300ng of template DNA Q5 High Fidelity 2X Master Mix, 0.2 μM VR and VF2 and water up to 20 μL A positive control was run using an RFP construct in psb1C3. A negative control was run using MilliQ water. Samples were run in the thermocycler in the following conditions: 98C for 30 seconds 32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds 72C for 120 seconds 4C Hold All PCR's were run on a 1% agarose gel containing 1.5 μL of ethidium bromide
WellSample
12 Log Purple DNA Ladder
3PCR1(MP1)
4PCR2(MP2)
5PCR3(MP3)
6PCR4(MP4)
7PCR5(MP5)
8PCR6(MP6)
9Positive Control
10Negative Control

Notes:



Stop: 5:30PM


Next:


TBD

14 September 2017

Ryan Baumann

Start: 2:00PM


PCR and Gel of 13 September 2017 minipreps


Purpose: To identify if Trg_PhoR and BBa_K1618037 Terminator are properly assembled in the UAP

Protocol:


PCR's of 9/13/17 MP1 (9/14/17 PCR1), MP2 (9/14/17 PCR2), and MP3 (9/14/17 PCR3), and an RFP positive control (9/14/17 PCR4) were run with the following conditions:
ComponentVolume
Taq 2X Master Mix10 μL
VR (10μM)0.4 μL
VF2 (10μL)0.4 μL
Samples0.7 μL
MilliQ8.5 μL
Total20 μL
PCR’s were run using taq2kbp program following taq 2x master mix protocol. Annealing temperatures calculated using IDT’s annealing tempature protocoL All Colony PCR’s were run on a 1% agarose gel containing 2.0 μL ethidium bromide. Gel Layout:
LaneComponent
12 Log Purple DNA Ladder
39/14/17 PCR1
59/14/17 PCR2
79/14/17 PCR3
99/14/17 PCR4

Notes:



Stop: 6:00PM


Next:


Round two of golden gate assemblies.

6 September 2017

Ryan Baumann

Start: 5:00PM


Golden Gate Assembly of BBa_K1618037 Terminator into UAP and Transformations


Purpose: To assemble plant terminator into the universale acceptor plasmid using BsmBI Type IIS endonuclease and transform on to chloramphenicol blue white screening plates. To transform amilCP chromoprotein (BBa_K592009) and eforRed Chromoprotein (BBa_K592012) on to chloramphenicol plates.

Protocol:


Reaction setup:
UAP0.20 μL (~60 ng)
BBa_K16180375 μL (~50 ng)
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ Water10.8 μL
20 μL
Samples Ran: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C 2 μL of the Terminator assembly, 1 μL of AmilCP, and 1 μL of eforRed were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol. The 1% and 10% dilutions were plated for each. AmilCP and eforRed were transformed on to chloramphenicol plates. The terminator assembly was plated on Chlor blue-white screening plates.

Stop: 10:30pm


Products:


LabelSourceDescription
9/6/17 AmilCP2017 Kit Plate 1 Well 19EamilCP resuspended from kit plate
9/6/17 eforRed2017 Kit Plate 7 Well 15IeforRed resuspended from kit plate

Next:


Check for colony growth. Plasmid mini prep desired colonies.

2 September 2017

Ryan Baumann

Start: Colony PCR and Gel of 8/31/17 Transformants


Confirm colonies with properly assembled plant parts with promoter and terminator


Purpose: To find properly assembled plasmids of PhoB, PhoR, and TNTr3 with promoter and terminator within alpha 1 acceptor plasmid.

Protocol:


20uL Colony PCR's were run for 7 PhoB colonies, 7 TNTr3 colonies, and 6 PhoR colonies. Sample colony PCR
ComponentVolume
Taq 2x Master Mix10 μL
VR0.8 μL
VF20.8 μL
MilliQ8.4 μL
Colonies 1-20
PCR's were run using taq2kbp program following taq 2x master mix protocol with annealing temperatures calculated using IDT's annealing tempature protocol. All Colony PCR's were run on a 1% agarose gel containing 1.5 μL ethidium bromide. Gel Layout
Lane(s)Sample
12 Log Purple Ladder
3-9PhoB colonies 1-7
10-12TNTr3 COlonies 1-3
132 Log Purple Ladder
15-18TNTR3 4-7
19-24PhoR colonies 1-6

Notes:



Stop:


Next:


Troubleshoot gel results.

1 September 2017

Benjamin Bleitz

Start: 1:00 pm


Preparation of Arabidopsis growth medium


Purpose: to prepare a proper growth medium for Arabidopsis in a laboratory setting

Protocol:


ComponentAmount
Murashige and Skoog salts2.15 g
1% Sucrose5 ml
0.05% MES0.25 ml
MilliQ H2Ototal of 500 ml
KOH Until pH adjusted to 5.7
Sucrose, MES and final solution all Autoclaved for 30 minutes on liquid cycle Plates poured

Stop: 3:30


Results:


18 plates prepared for plant growth

Products:


18 MS Mediums

Next:


Test growth mediums

31 August 2017

Ryan Baumann

Start: 3:00 PM


Addition of Promoter and Terminator to PhoR, PhoB, and TNTR3


Purpose: Addition of BBa_K1467101 Promoter and BBa_K1618037 Terminator to PhoR, PhoB, and TNTR3 into BBa_P10501 Alpha 1 acceptor plasmid via golden gate assembly using NEB golden gate assembly kit.

Protocol:


20 uL Reactions were set up following NEB golden gate assembly mix using 75ng of BBa_P10501 as the destination plasmid and 100ng of each insert. All samples were run at 37C for 1 hour followed by 55C for 5 minutes. 1 uL of each assembly were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol. 20% and 2% dilutions were then plated on Kanamysin selective blue white screening plates.

Stop: 7:00PM


Results:


Growth of white colonies was seen from assembly transformation.

Next:


Colony PCR from transformation plates to confirm proper assembly

23 August 2017

Ryan Baumann

Start: 3:00 PM


PCR of August 18th plasmid minipreps with VR and VF2 and gel extraction


Purpose: To verify correct assembly of fragments in the Universal Acceptor Plasmid and extract correct bands from gel

Protocol:


~100 ng of purified template DNA was amplified following the iGEM Lab Manual 2.5 PCR Protocol using Taq 2x MM, VR, VF2, and MilliQ H2O. A positive control was run using linearized Psb1T3 backbone. A negative control as run with a water template. Sample Reaction:
SampleVolume
Taq 2X Master Mix 12.5 μL
VR1 μL
VF21 uL
Plant Pho (100ng/1uL)1 uL
Water 9.5 μL
Total25 μL
Samples were run in thermocycler using Taq 2X master mix recommend protocol with appropriate annealing temperature for VR and VF2. All PCR's were run in a 1% agarose gel containing 2 μL ethidium bromide. Gel Layout:
LaneSample
12 Log Purple DNA ladder
3Negative Control
4TNTR3
5Plant Pho
6Positive Control
7PhoB
8PhoR
Bands containing Assembled TNTR3 (~1100BP), Plant Pho (~450BP), PhoB (~1100BP), and PhoR (~1800 BP) were cut from the gel and DNA was purified using Amicon Ultra-DA Kit for DNA extraction from agarose gel.

Notes:



Stop: 5:06 PM


Products:


Label Source Description
8/23/17 Plant Pho8/18/17 plant Pho MiniprepExtracted plant pho DNA from gel
8/23/17 Pho R8/18/17 PhoR miniprepextracted PhoR from gel
8/23/17 Pho B8/18/17 PhoB miniprepExtracted PhoB from Gel
8/23/17 TNTR38/18/17 tntr3 miniprepExtracted TNTr3 from gel

Next:



18 August 2017

Ryan Baumann

Start: 11:00am


Plasmid Miniprep of colonies from 8/9/17 COlony PCR's


Purpose: To isolate plasmids from colonies with correctly assembled PhoB, OhoR, Plant pho, and TNTR3

Protocol:


Colonies that showed positive bands from PCR were innoculated into broth cultures with chloramphenicol and grown up overnight. Plasmid DNA was purified using the MST iGEM Lab Manual 2.5 Kitless miniprep protocol and suspended in 40uL of TE buffer. 2uL of sample of was run on Thermoscientific Nanodrop 1000. Nanodrop results:
SampleConcentration (ng/uL)260nm/280nm
PhoB263.21.84
PhoR98.6 1.80
Plant Pho99.81.86
TNTr3236.01.82

Stop: 1:40PM


Products:


LabelSourceDescription
8/18/17 MP1PhoB Colonies from 6/26/17 TransformationPhoB in the UAP
8/18/17 MP2PhoR from 6/26/17 TransformationPhoR in the UAP
8/18/17 MP3Plant Pho from 6/26/17 TransformationPlant Pho in the UAP
8/18/17 MP4TNTR3 from 6/26/17 TransformationTNTr3 in the UAP

Next:


PCR with VR and VF2 and extract band containing correctly assembled plasmid for sequencing. If correctly assembled, perform golden gate assembly to add Promoter and terminator to each plant part.

9 August 2017 to 10 August 2017

Ben Bleitz

Start: 2:00 PM of 9 August 2017


PCR and Gels of 18 Colonies from the PhoR Plate


Purpose: Correctly identify assembled of plasmid containing PhoR in universal acceptor

Protocol:


PCRs were conducted for marked colonies 1-18 on PhoR
ComponentVolume
Taq 2x Master Mix10μl
VR 0.8μl
VF20.8μl
MilliQ8.4μl
Colonies 1-18
Samples were run through the Taq2bkp program and then frozen for the next day All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide with 2 Log Purple DNA ladder, the following day.

Stop: 1:00 PM of 10 August 2017


Results:



Next:


Inoculate culture from appropriate culture

8 August 2017

Ryan Baumann

Start: 4:00PM


Colony PCR of 6/26/17 TNTR3 transformation


Purpose: To identify correctly assembled plasmid containing TNTr3 in the universal acceptor plasmid 20 μL Sample reaction
Componentvolume
Taq 2X Master Mix10 μL
VR0.8 μL
VF20.8 μL
MilliQ8.4 μL
Colonies 1-16
A positive Control was conducted and run
Componentvolume
Taq 2X Master Mix10 μL
VR0.8 μL
VF20.8 μL
MilliQ7.4 μL
7/24/17 K6080021 uL
A negative control PCR was conducted and run
Componentvolume
Taq 2X Master Mix10 μL
VR0.8 μL
VF20.8 μL
MilliQ8.4 μL
20 μL
All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide wit 2 Log Purple DNA ladder.

Protocol:


PCR's of 16 colonies were run with VR and VF2 primers, Taq 2x Master mix, and MilliQ Water

Notes:



Stop: 9:00 PM


Results:


TBD

Next:


Examine Gel and find if colonies match correctly assembled plasmid

3 August 2017

Ben Bleitz

Start: 2:00


Running Gel of Plant Pho, PhoB, PhoR, TNTR3


Purpose: Running Gels of repeat PCR

Protocol:


LaneComponent
110 uL NEB 2 Log Purple DNA ladder
310 uL 7/27/17 PCR 5
510 uL 7/27/17 PCR 1
610 uL 7/27/17 PCR 2
710 uL 7/27/17 PCR 3
8
910 uL 7/24/17 K608002 (+ Control)
1010 uL MilliQ Water (- Control)

Stop: 5:00


Results:


Somehow managed to repeat mistakes from last week. Will repeat, for a third and hopefully final time for last week.

Next:


DONT MESS UP

2 August 2017

Ben Bleitz

Start: 1:00 pm


PCR of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)


Purpose: Repeat of previous PCR (and eventual gel) from 27 June 2017. Gel scheduled to be completed following day (3 August 2017)

Protocol:


Plant Pho PCR:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
7/24/17 Plant Pho3 μL
MilliQ 7.5 μL
Total25 μL
Pho B Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP11.5 μL
MilliQ 9 μL
Total25 μL
TNTR3 Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP62.0 μL
MilliQ 8.5 μL
Total25 μL
PhoR Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP21.5 μL
MilliQ 9 μL
Total25 μL
7/25/17 L1
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
7/25/17 L15 μL
MilliQ 5.5 μL
Total25 μL

Notes:


PCR was completed. Gel will be ran following day

Stop: 4:20 pm (#Blazeit)


Results:


PCRs frozen after thermocycler completed. Will be determined upon gel electrophoresis completion on the following day.

Products:


Label Source Description
PCR 1 7/24/17 Plant Pho7/24/17 Plant Pho amplified with VR and VF2
PCR 26/29/17 MPI6/29/17 MPI amplified with VR and VF2
PCR36/29/17 MP66/29/17 amplified with VR and VF2
PCR46/29/17 MP26/29/17 amplified with VR and VF2
PCR57/25/17 L17/25/17 L1 amplified with VR and VF2

Next:


Run gel

27 June 2017

Erin Nischwitz, Ben Bleitz

Start: 11:00 am


PCR and gels of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)


Purpose: To verify assembly of these constructs

Protocol:


Five PCR's were ran with VR and VF2 using Q5 2x Master mix Protocol Plant Pho PCR:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
7/24/17 Plant Pho3 μL
MilliQ 7.5 μL
Total25 μL
Pho B Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP11.5 μL
MilliQ 9 μL
Total25 μL
TNTR3 Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP62.0 μL
MilliQ 8.5 μL
Total25 μL
PhoR Pcr:
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
6/29/17 MP21.5 μL
MilliQ 9 μL
Total25 μL
7/25/17 L1
ComponentVolume
Q5 2x Master Mix12.5 μL
VR1μL
VF21 μL
7/25/17 L15 μL
MilliQ 5.5 μL
Total25 μL
A 1% agarose gel was ran containing all five PCRs, a positive control, and a Negative control
LaneComponent
110 uL NEB 2 Log Purple DNA ladder
310 uL 7/27/17 PCR 5
510 uL 7/27/17 PCR 1
610 uL 7/27/17 PCR 2
710 uL 7/27/17 PCR 3
810 uL 7/27/17 PCR 4
910 uL 7/24/17 K608002 (+ Control)
1010 uL MilliQ Water (- Control)

Notes:


-Accidentally used circular DNA as a positive control, so do not expect a valid + Control

Stop: 3:30


Results:


TBD

Products:


Label Source Description
PCR 1 7/24/17 Plant Pho7/24/17 Plant Pho amplified with VR and VF2
PCR 26/29/17 MPI6/29/17 MPI amplified with VR and VF2
PCR36/29/17 MP66/29/17 amplified with VR and VF2
PCR46/29/17 MP26/29/17 amplified with VR and VF2
PCR57/25/17 L17/25/17 L1 amplified with VR and VF2

Next:


Trouble Shoot

24 July 2017

Ryan Baumann

Start: 11:00 AM


Plasmid Miniprep of 7/21/17 Transformations


Purpose: Prepare plasmids for future use

Protocol:


Kitless miniprep protocol from igem lab manual version 2.5 Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 12:40PM


Results:


Samples tested on Thermoscientific Nanodrop 1000
LabelDNA Conc.260/280
BBa_B0015893.5 ng/uL1.95
BBa_K608002573.5 ng/uL1.88
Plant Pho165.7 ng/uL1.93
Skype Backpack

Products:


LabelSourceDescription
7/24/17 B00152017 Kit Plate 3 well 3FB0015 Terminator in psb1c3
7/24/17 Plant PhoPlant Pho top and bottom strandPlant pho in UA plasmid
7/24/17 K6080022017 Kit Plate 1 well 3OK608002 Promoter and RBS in psb1c3

Next:



25/June/2017

Ben Bleitz

Start: 1:30


Digestion and Ligation of BBa_K608002 and TNTR3_E.Coli


Purpose: To prepare DNA Samples for Transformations

Protocol:


Two double digestions were performed BBa_K608002 Double digestion
Component Volume
ECORI Enzyme1 μl
SpeI Enzyme1 μl
K608002 Insert3.5 μl
Tango Buffer 2.5 μl
MilliQ H2O17 μl
TNT_E.Coli Double Digestion
Component Volume
ECORI Enzyme1 μl
Xbal Enzyme 1 μl
TNTR3_E.Coli Vector5 μl
Tango Buffer2.5 μl
MilliQ H2O15.5 μl
Products were 25/7/2017 d1 and 25/7/2017 d2 Ligation was then performed
ComponentVolume
25/7/2017 d1 (insert)1.5 μl
25/7/2017 d2 (vector)2 μl
T4 Ligase Buffer2 μl
T4 Ligase1 μl
13.5 MilliQ H2O13.5

Stop: 5:30


Products:


Label SourceDescription
25/7/2017 d1BBa_K608002K608002 cut with ECORI & SPeI
25/7/2017 d2TNTR3_E.ColiTNTR3_E.Coli cut with ECORI & Xbal
25/7/2017 L1d1 and d2DNA formed from d1 insert and d2 vector

Next:


PCR with VR and VF 2 to confirm products

20/7/2017

Ben Bleitz

Start: 2:30


Bacterial promoter/ terminator and plant pho Transformations


Purpose: Transform components for later prep and kit plate

Protocol:


Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.
TransformantDNA VolumePlate Dilutions
Plant Pho1μl2 and 20%
BBa_B00151μl2 and 20%
BBa_K6080021μl2 and 20%

Stop: 5:30


Results:


All samples in incubation. Results will be noted at the end of 24 hour incubation period

Products:


LabelSourceDescription
BBa_B00152017 Kit Plate 3 well 3FTransformed on chlorophenicol plate
BBa_K608002 2017 Kit Plate 1 well 3OTransformed on chlorophenicol plate
Plant Pho Plant Pho top and bottom strandTransformed on Blue/White chlorophenicol plate

Next:


Inoculate in broth culture with appropriate antibiotic for future miniprep.

10 July 2017

Ryan Baumann

Start: 12:30


Golden Gate assembly of Plant Pho Top and Bottom strand into UA plasmid


Purpose: To assemble PlantPho Oligos into UAP to prepare for assembly of Transcriptional Unit

Protocol:


UA MP1 (100ng)0.3 μL
PlantPho Top Strand (100 ng)1 μL
PlantPho Bottom Strand (100 ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O13.7 μL
20 μL
Ran at: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C

Stop: 5:00


Products:


LabelSourceDescription
7/10/17Plant Pho Top & Bottom Strand OligosPlant Pho Oligos cut with BsmBI and Ligated into UAP

Next:


Transform on to Chlor Blue white Screening Plates

29 June 2017

Ryan Baumann, Ben Bleitz

Start: 1:30


Miniprep of 6/26/17 transformations


Purpose: Prepare plasmids for future use

Protocol:


Kitless miniprep protocol from igem lab manual version 2.5 Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 3:30


Results:


Samples tested on Thermoscientific Nanodrop 1000
LabelDNA Concentration (ng/μl)260/280
MP1412.71.84
MP2399.61.83
MP3290.21.82
MP4722.41.80
MP5406.41.81
MP6307.41.84

Products:


Label SourceDescription
6/29/17 MP1PhoB-VP64 Transformation platePhoB-VP64 transformed into UA Plasmid
6/29/17 MP2Trg-PhoR Transformation plateTrg-PhoR transformed into UA Plasmid
6/29/17 MP3Golden Braid Omega 1 Plasmid Transformation plateGolden Braid Omega 1 plasmid
6/29/17 MP4Golden Braid Omega 2 Plasmid Transformation plateGolden Braid Omega 2 plasmid
6/29/17 MP5TNTR3 E. Coli Transformation plateTNTR3 E. Coli in PSB1C3 backbone
6/29/17 MP6TNTR3 Transformation plateTNTR3 transformed into UA plasmid

Next:


TBD

6/26/2017

Ryan Baumann, Ben Bleitz, Erin Nischwitz

Start: 5:00 pm


Chemical Transformations


Purpose: To transform PhoB-VP64, Trg_PhoR, Plant Pho, and TNTR3 on to chloramphenicol blue white screening plates. To transform TNTR3_E Coli (6/22/17 L1) on to chloramphenicol plate To transform Golden Braid Omega 1 and Omega =1 plasmids on to Streptomycin plates.

Protocol:


Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.
TransformantDNA volumePlate Dillutions
TNTR31 μL2% and 20%
Trg_PhoR1 μL2% and 20%
PhoB_VP641 μL2% and 20%
Plant Pho2 μL2% and 20%
6/22/17 L1 (TNTR3 E coli)3 μL5% and 50%
Omega 11 μL2% and 20%
Omega Two1 μL2% and 20%

Stop: 7:45


Next:


Check for colonies and inoculate cultures

6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm


Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.


Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:


Trg_PhoR-Gblock:
UA MP1 (100ng) 0.30 μL
Trg_PhoR (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
PhoB_VP64-gblock
UA MP1 (100ng) 0.30 μL
PhoB_VP64 (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
TNT_R3-gblock
UA MP1 (100ng) 0.30 μL
TNT_R3 (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
PlantPho Oligos Top and Bottom Strands
UA MP1 (100ng)0.30 μL
PlantPho Top Strand (100 ug)1μL
PlantPho Bottom Strand (100 ug)1μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1μL
BsmBI1μL
MilliQ H2O13.7 μL
20 μL
All Samples Ran: - 20 seconds @ 37°C, - (3 minutes @ 37°C, 4 minutes @ 16°C) X26 - 5 minutes @ 50°C - 5 minutes @ 80°C - 5 minutes @ 16°C

Notes:


100 μg of top and bottom strand oligos were accidentally used instead of 100ng.

Stop: 4:45 PM


Products:


LabelSource Description
6/26 TNTR3TNTR3-GBlockTNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant PhoPlant Pho Top & Bottom Strand OligosPlant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoRTrg_PhoR-GblockTrg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoBPhoB_VP64 GblockPhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

Next:


Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance

6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm


Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.


Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:


Trg_PhoR-Gblock:
UA MP1 (100ng) 0.30 μL
Trg_PhoR (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
PhoB_VP64-gblock
UA MP1 (100ng) 0.30 μL
PhoB_VP64 (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
TNT_R3-gblock
UA MP1 (100ng) 0.30 μL
TNT_R3 (100ng)1 μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1 μL
BsmBI1 μL
MilliQ H2O14.7 μL
20 μL
PlantPho Oligos Top and Bottom Strands
UA MP1 (100ng)0.30 μL
PlantPho Top Strand (100ng)1μL
PlantPho Bottom Strand (100ng)1μL
10X T4 DNA Ligase Buffer2 μL
T4 DNA Ligase1μL
BsmBI1μL
MilliQ H2O13.7 μL
20 μL
All Samples Ran: - 20 seconds @ 37°C, - (3 minutes @ 37°C, 4 minutes @ 16°C) X26 - 5 minutes @ 50°C - 5 minutes @ 80°C - 5 minutes @ 16°C

Stop: 4:45 PM


Products:


LabelSource Description
6/26 TNTR3TNTR3-GBlockTNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant PhoPlant Pho Top & Bottom Strand OligosPlant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoRTrg_PhoR-GblockTrg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoBPhoB_VP64 GblockPhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

Next:


Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance

23/6/17

Ben Bleitz

Start: 2:50pm


Ligation of PsbIC3 (Vector) and 6/21/17 DI (insert)


Purpose: To create recombinant DNA for transformation

Protocol:


T4 Ligase Buffer2μL
PsbIC3 (Vector)4μL
6/21/17 DI (Insert)3μL
MilliQ H2O10μL
T4 DNA Ligase1μL
Thermal Cycler 16°C for 30 minutes 80°C for 20 minutes

Stop: 4:00pm


Results:


6/23/17 L1

Next:


E. Coli Transformation

6/21/2017

Ben Bleitz

Start: 2:00


Double Digestion of TNT R3_E.Coli


Purpose: To prepare TNT R3_E.Coli for submission into the registry

Protocol:


Volume Component
2.5 μL10X Tango Buffer
1 μLECORI
1 μLPSTI
15 μLTNTR3_E.Coli (10 ng/μL)
5.5 μLMilliQ H2O
25 μLin Total
Ran @ 37° C for 1 hour Heat kill @ 80°C for 30 minutes

Stop: 4:00 PM


Products:


LabelSourceDescription
6/21/17 D1TNTR3_E.Coli TNTR3_E.Coli cut with ECORI and PSTI

Next:


Ligate into PsbIC3

×

Loading ...