Team:Rice/Medal Requirements
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Week of 09/04 (post-Harvey)
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-04 to 2017-09-08
Monday, 9/4/17
People present
Lab Silberg, Keck 201
Goal Golden Gate Assembly PgntK, ChrB, sbp
Time in Lab 3pm - 5:30pm
A | B | C | D | |
1 | ChrB | sbp | ||
2 | Reagent | Volume (uL) | Reagent | Volume (uL) |
3 | p439 (10x) | 1.6 | p439 (10x) | 1.6 |
4 | Con L4 (505) 10x | 1.9 | 459 | 0.2 |
5 | ChrB (10x) | 1.8 | sbp | 0.48 |
6 | Con RE (520) 10x | 1.8 | 462 | 0.22 |
7 | 520 (10x) | 1.8 | ||
8 | ||||
9 | BsaI | 0.5 | BsaI | 0.5 |
10 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 |
11 | T4 Buffer | 1.5 | T4 Buffer | 1.5 |
12 | 10X BSA | 1.5 | 10X BSA | 1.5 |
13 | H20 | 3.9 | H20 | 6.7 |
ChrB and sbp Cassette Assembly
PgntK Part Plasmid Assembly
A | B | C | D | E | F | G | H | I | J | |
1 | Volume of Components to Add by Assembly Reaction (volume in uL) | |||||||||
2 | Insert # | |||||||||
3 | Esp3I | T4 DNA Ligase | 10x T4 Ligase Buffer | 10X BSA | 1 | 2 | Plasmid | dH2O | TOTAL VOLUME | |
4 | Pgntk | 0.5 | 0.5 | 1.5 | 1.5 | 0.34 | 0.21 | 10.45 | 15 | |
Repeated Golden Gate Assembly
Week of 09/04 (post-Harvey)
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-04 to 2017-09-08
Tuesday, 9/5/17
PgntK, ChrR6 BioBrick characterization - cassette assembly**
** postponed until we confirm the RBSs to use
A | B | C | D | E | F | |
1 | ChrR6 c1 | ChrR6 c2 | Pcon | |||
2 | Reagent | Volume (uL) | Reagent | Volume (uL) | Reagent | Volume (uL) |
3 | 439 10x | 1.6 | 439 | 1.6 | 439 | 1.6 |
4 | 501 (conLS) | 0.25 | 522 (OpconL2) | 0.24 | 501 (conLS) | 0.25 |
5 | 476 (Pcon)(10x) | 1.9 | 457 (RBS) | 0.26 | Pcon(476) 10x | 1.9 |
6 | 459 (RBS) | 0.2 | 453 (yfp) 10x | 1.4 | RBS(457) | 0.26 |
7 | ChrR6 | 0.38 | 463 (terminator) | 0.21 | 453 (yfp) 10x | 1.4 |
8 | 531 (OpconL2) 10x | 1.9 | 520 (conRE) 10x | 1.8 | terminator (463) | 0.21 |
9 | conR1 (511) 10x | 1.4 | ||||
10 | ||||||
11 | BsaI | 0.5 | BsaI | 0.5 | BsaI | 0.5 |
12 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 |
13 | T4 Buffer | 1.5 | T4 Buffer | 1.5 | T4 Buffer | 1.5 |
14 | 10X BSA | 1.5 | 10X BSA | 1.5 | 10X BSA | 1.5 |
15 | H20 | 6.37 | H20 | 7.09 | H20 | 5.58 |
Cassette Assembly REaction Set UP
Golden Gate Assemblies Transformation
Transform PgntK old*, PgntK new, sbp, ChRB following heat shock protocol.
* PgntK old is from the previous assembly (antediluvian), discovered in a fridge.
A | B | C | D | E | F | G | H | I | |
1 | sample | concentration (ng/uL) | amount to use in reaction (of 10 x dilution) | Phusion GC buffer | dNTPs | Fwd Primer | Rev Primer | Q5 DNA polymerase | H2O |
2 | ChrB2 | 111.3 | 0.9 | 10 | 1 | 2 | 2 | 0.5 | 33.6 |
3 | ChrB3 | 113.0 | 0.9 | 10 | 1 | 2 | 2 | 0.5 | 33.6 |
ChrB PCR
Results:
Well 1: 2 Log ladder
Well 2: ChrB2
Well 3: ChrB 3
Expected fragment length: 1116, which is about the size of the bands observed
Week of 09/04 (post-Harvey)
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-04 to 2017-09-08
Wednesday, 9/6/17
PgtnK and sbp Colony pcr
Chromium Remediation Circuit Multigene Assembly
A | B | |
1 | Multigene assembly | |
2 | Reagent | Volume (uL) |
3 | 549 | 0.21 |
4 | 539 | 0.22 |
5 | KS 2 cassette | 1.94 |
6 | ChrR6 cassette | 1.46 |
7 | yfp cassette | 0.95 |
8 | LacI cassette | 0.99 |
9 | ChrB 2 cassette | 0.74 |
10 | 543 | 0.38 |
11 | ||
12 | Esp3I | 0.5 |
13 | T4 DNA Ligase | 0.5 |
14 | T4 Buffer | 1.5 |
15 | 10X BSA | 1.5 |
16 | H20 | 4.11 |
Table 2: Multigene Assembly
sbp Assmebly
Since no colonies were observed for the transformation (neither green nor white), I concluded that I did not add the correct amount
A | B | |
1 | sbp | |
2 | Reagent | Volume (uL) |
3 | p439 (10x) | 1.6 |
4 | 521 | 0.22 |
5 | 459 | 0.2 |
6 | sbp | 0.48 |
7 | 462 | 0.22 |
8 | 520 (10x) | 1.8 |
9 | ||
10 | BsaI | 0.5 |
11 | T4 DNA Ligase | 0.5 |
12 | T4 Buffer | 1.5 |
13 | 10X BSA | 1.5 |
14 | H20 | 6.48 |
sbp Assmebly
Week of 09/04 (post-Harvey)
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-04 to 2017-09-08
Thursday, 9/7/17
People present Catherine
Lab Silberg, Keck 201
Goal Perform colony PCR on Pgtnk part plasmid transformation and sbp cassette transformation
Time in Lab 9am - 10:40am
Observations
Retrieved transformation plates from the incubator. See Figure 1 below. Because no colonies were observed on the sbp part plasmid plates, I was not able to perform colony PCR. (I returned sbp and chrb plates to incubator and put the Pgtnk "old" and "new" plates into the igem box in the fridge.)
Procedure: colony PCR on Pgtnk part plasmid colonies
1.
Picked 5 white and 1 green colony from the "Pgtnk new 9/6" plate and resuspended each in 5 ul of LB + Chl34.
2.
Prepared a PCR master mix (see Table 1 below) and distributed 24 ul of the MM to each of 6 PCR reaction tubes. Then, added 1 ul of resuspended colony into the corresponding tubes as the template DNA
a.
Once the colonies were added to the PCR mix, I put the resuspended colonies into the incubator in a ziploc
3.
Put the PCR reactions into the thermocycler with the conditions detailed in Table 2 below.
4.
I prepared a 1% Agarose Gel-Green Gel with 12 wells and loaded the samples as well as a 2-log ladder into the gel with Purple Loading dye according to the layout in Figure 2 below:
A | B | C | |
1 | Single colony PCR Reaction | Master Mix (6.6 Reactions) | |
2 | Template DNA (ul) | 1 | --- |
3 | F Primer (B17) (ul) | 1.25 | 8.25 |
4 | R Primer (B18) (ul) | 1.25 | 8.25 |
5 | dNTPs (ul) | 0.5 | 3.3 |
6 | 5X Q5 Reaction Buffer (ul) | 5 | 33 |
7 | Q5 High Fidelity DNA Polymerase (ul) | 0.25 | 1.65 |
8 | H20 (ul) | 15.75 | 103.95 |
9 | Total | 25 |
Table 1: Colony PCR on Pgntk part plasmid
6 reactions total; prepared 6.6 reactions worth of master mix to have some excess
Q5 protocol: https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491
A | B | C | |
1 | STEP | TEMP | TIME |
2 | Initial Denaturation | 98°C | 30 seconds |
3 | 35 Cycles | 98°C | 10 seconds |
4 | 71°C | 30 seconds | |
5 | 72°C | 30 seconds (255 bp) | |
6 | Final Extension | 72°C | 2 minutes |
7 | Hold | 4–10°C | infinite |
Table 2: Thermocycling for Pgntk colony PCR
Procedure: Liquid Culture of Pgntk (new) Colonies
1.
Started liquid cultures of colonies 3 and 4 (as seen in Figure 2 above) by adding 4ul of resuspended colony to 4 ml of LB+Chl34 in falcon tubes
2.
placed falcon tubes with lids loosely capped into the 37C, 250 rpm incubator at 4:26pm
Procedure: Transform Repeated sbp assembly
1.
Recovered repeated sbp part plasmid assembly from thermocycler (Anna performed assembly this afternoon)
2.
Pre-warmed (2) LB+Chl34 plates in the 37C inubator
3.
Thawed aliquot of heat-shock competent MG1655 cells on ice
4.
added 1ul of the sbp assembly reaction to the thawed cells and flicked to mix
5.
Incubated mixture on ice for 30 min
6.
Transferred mixture to 42C heat block for 45 seconds then transferred back to ice for 2 minutes
7.
Added 1 mL of SOC to the mixture and incubated at 37C and 250 rpm for 1 hour
8.
Plated 200ul of the transformation onto 1 plate and 800ul onto the 2nd plate at 6:11 pm
9.
Incubated overnight
Week of 09/04 (post-Harvey)
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-04 to 2017-09-08
Friday, 9/8/17
A | B | C | |
1 | Single colony PCR Reaction | Master Mix (6.6 Reactions) | |
2 | Template DNA (ul) | 1 | --- |
3 | F Primer (B17) (ul) | 1.25 | 8.25 |
4 | R Primer (B18) (ul) | 1.25 | 8.25 |
5 | dNTPs (ul) | 0.5 | 3.3 |
6 | 5X Q5 Reaction Buffer (ul) | 5 | 33 |
7 | Q5 High Fidelity DNA Polymerase (ul) | 0.25 | 1.65 |
8 | H20 (ul) | 15.75 | 103.95 |
9 | Total | 25 |
Table 1: Colony PCR on sbp
A | B | C | |
1 | STEP | TEMP | TIME |
2 | Initial Denaturation | 98°C | 30 seconds |
3 | 35 Cycles | 98°C | 10 seconds |
4 | 71°C | 30 seconds | |
5 | 72°C | 34 seconds (1.132 kb fragment) | |
6 | Final Extension | 72°C | 2 minutes |
7 | Hold | 4–10°C | infinite |
Table 2: Thermocycling Conditions for sbp colony PCR
A | B | |
1 | NAME | ng/uL |
2 | Pgntk miniprep colony 3 | 180.3 |
3 | Pgntk miniprep colony 4 | 166.6 |
Table 3: Pgntk part plasmid miniprep specs
The previously gel purified sbp and chrR6 part plasmid digest samples had a very low concentration (5.2 and 3.3 ng/ul respectively), so we decided to repeat a digestion with a larger volume of sample. Below is the procedure for a 50 ul digestion reaction (originally we did 20 ul reactions).
Incubated at 37C for 1 hour and then ran on a 1% agarose gel green gel
A | B | C | D | |
1 | sbp part plasmid (162 ng/uL) | ChrR6 part plasmid (161.8 ng/uL) | chrR6 cassette (51.7 ng/ul) | |
2 | DNA (ul for ~1 ug) | 6.17 | 6.17 | 19.34 |
3 | CutSmart 10X Buffer (ul) | 5 | 5 | 5.00 |
4 | EcoRI-HF (ul) | 2.5 | 2.5 | 2.50 |
5 | PstI-HF (ul) | 2.5 | 2.5 | 2.50 |
6 | H2O (ul) | 33.83 | 33.83 | 20.66 |
7 | Total | 50 | 50 | 50 |
Double Digest of sbp and chrR6 part plasmids
A | B | C | D | E | F | G | H | |
1 | sample | gel volume (ul) | ADB (ul) | concentration (ng/uL) | A260 | A280 | 260/280 | 260/230 |
2 | chrR6 part plasmid, digested | 170 | 510 | 6.2 | 0.124 | 0.048 | 2.56 | 0.31 |
3 | sbp part plasmid, digested | 260 | 780 | 11.8 | 0.237 | 0.107 | 2.2 | 0.46 |
Table 1: Gel Purification of sbp and chrR6 for ligation into BioBrick Vector
Performed 2 ligations according to procedure here: http://parts.igem.org/Help:Protocols/3A_Assembly
used 3ul of the digested samples and 2 ul of the digested backbone
A | B | |
1 | sample | concentration (ng/uL) |
2 | chrB 2 cassette | 117.6 |
3 | lacI 1 cassette | 77.0 |
4 | KS 2 cassette | 43.6 |
5 | yfp cassette | 77.3 |
6 | chrR6 | 56 |
Confirming concentrations of cassettes with nanodrop
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Monday, 9/11/17
People present Anna
Lab Silberg, Keck 201
Goal Transformations of samples from Friday
Transformed five cassettes, Chr6 cassettes 1 and 2, Pcon cassette for BioBrick characterization using heat shock and multigene assembly with electroporation
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Tuesday, 9/12/17
A | B | C | |
1 | Single colony PCR Reaction | Master Mix (11.6 Reactions) | |
2 | Template DNA (ul) | 1 | --- |
3 | F Primer (B17) (ul) | 1.25 | 14.5 |
4 | R Primer (B18) (ul) | 1.25 | 14.5 |
5 | dNTPs (ul) | 0.5 | 5.8 |
6 | 5X Q5 Reaction Buffer (ul) | 5 | 58 |
7 | Q5 High Fidelity DNA Polymerase (ul) | 0.25 | 2.9 |
8 | H20 (ul) | 15.75 | 182.7 |
9 | Total | 25 |
Table 1: Colony PCR on sbp cassette and multigene assembly
5 sbp colonies and 6 multigene colonies
A | B | C | D | |
1 | STEP | TEMP | TIME for MULTIGENE | TIME for sbp cassette |
2 | Initial Denaturation | 98°C | 30 seconds | 30 seconds |
3 | 35 Cycles | 98°C | 10 seconds | 10 seconds |
4 | 65°C | 30 seconds | 30 seconds | |
5 | 72°C | 2 min 33 seconds (5.078 kb fragment) | 37 sec | |
6 | Final Extension | 72°C | 2 minutes | 2 minutes |
7 | Hold | 4–10°C | infinite | infinite |
Table 2: Thermocycling Conditions for colony PCR
Made liquid culture of sbp colonies 1 and 3
Transformations
Performed the transformations of sbp and ChrR6 Biobricks, c1, c2, and Pcon according to Protocol 4.
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Wednesday, 9/13/17
A | B | |
1 | NAME | ng/uL |
2 | lacI cassette | 224 |
3 | sbp 2 cassette | 627.1 |
4 | sbp 1 cassette | 996.2 |
5 | yfp cassette | 144.1 |
6 | chrB cassette | 114.1 |
7 | chrR6 cassette | 232.5 |
8 | KS cassette | 111.1 |
Miniprepped Cassette Samples
storred in a new box called "Cassettes starting 9/13/17"
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Thursday, 9/14/17
A | B | |
1 | NAME | ng/uL |
2 | sbp cassette | [on tube] |
3 | chrR6 cassette #1 | [on tube] |
4 | chrR6 cassette | [on tube] |
miniprepped cassette samples
A | B | C | D | E | F | G | H | |
1 | Sample | ng/ul | volume of sample for 500 ng (ul) | volume of primers (ul) | volume of water (ul) | primer ID | name on order | # on order |
2 | lacI cassette | 224 | 4.46 | 2.5 | 8.04 | B17 | lacI cass_fwd | 1 |
3 | sbp 2 cassette | 627.1 | 1.59 | 2.5 | 10.91 | B17 | sbp2 cass_fwd | 2 |
4 | sbp 1 cassette | 996.2 | 1.00 | 2.5 | 11.50 | B17 | sbp1 cass_fwd | 3 |
5 | yfp cassette | 144.1 | 6.94 | 2.5 | 5.56 | B17 | yfp cass_fwd | 4 |
6 | chrB cassette | 114.1 | 8.76 | 2.5 | 3.74 | B17 | chrB cass_fwd | 5 |
7 | chrR6 cassette | 232.5 | 4.30 | 2.5 | 8.20 | B17 | chrR6 cass_fwd | 6 |
8 | KS cassette | 111.1 | 9.00 | 2.5 | 3.50 | B17 | KS cass_fwd | 7 |
9 | lacI cassette | 224 | 4.46 | 2.5 | 8.04 | B18 | lacI cass_rev | 8 |
10 | sbp 2 cassette | 627.1 | 1.59 | 2.5 | 10.91 | B18 | sbp2 cass_rev | 9 |
11 | sbp 1 cassette | 996.2 | 1.00 | 2.5 | 11.50 | B18 | sbp1 cass_rev | 10 |
12 | yfp cassette | 144.1 | 6.94 | 2.5 | 5.56 | B18 | yfp cass_rev | 11 |
13 | chrB cassette | 114.1 | 8.76 | 2.5 | 3.74 | B18 | chrB cass_rev | 12 |
14 | chrR6 cassette | 232.5 | 4.30 | 2.5 | 8.20 | B18 | chrR6 cass_rev | 13 |
15 | KS cassette | 111.1 | 9.00 | 2.5 | 3.50 | B18 | KS cass_rev | 14 |
Sequencing Order
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Friday, 9/15/17
People present Albert, Anna
Lab Silberg, Keck 201
Goal Send samples for sequencing, prepare competent cells, assemble cys + PgntK cassette, assemble BioBrick test cassettes: Pcon_B, ChrR6#2_B and also Pgntk_B (_B for BioBrick to avoid confusion)
cys + PgntK cassette assembly
A | B | |
1 | cys + PgntK | |
2 | Reagent | Volume (µL) |
3 | p439 (10x dil) | 1.6 |
4 | 501 (LS) | 0.25 |
5 | PgntK | 0.29 |
6 | cysPUWA | 2.76 |
7 | 530 | 0.22 |
8 | ||
9 | BsaI | 0.5 |
10 | T4 DNA Ligase | 0.5 |
11 | T4 Buffer | 1.5 |
12 | 10X BSA | 1.5 |
13 | H20 | 5.88 |
Table1: cys + PgntK cassette assembly
A | B | C | D | E | F | |
1 | ChrR6 c2 | Pcon | PgntK | |||
2 | Reagent | Volume (uL) | Reagent | Volume (uL) | Reagent | Volume (uL) |
3 | 439 | 1.6 | 439 | 1.6 | 439 | 1.6 |
4 | 522 (OpconL2) | 0.24 | 501 (conLS) | 0.25 | 501 (conLS) | 0.25 |
5 | 457 (RBS) | 0.26 | Pcon(476) 10x | 1.9 | PgntK | 0.29 |
6 | 453 (yfp) 10x | 1.4 | RBS(457) | 0.26 | ||
7 | 463 (terminator) | 0.21 | 454 (mCherry) | 0.2 | 454 (mCherry) | 0.2 |
8 | 520 (conRE) 10x | 1.8 | terminator (463) | 0.21 | terminator (463) | 0.21 |
9 | conR1 (511) 10x | 1.4 | conR1 (511) 10x | 1.4 | ||
10 | ||||||
11 | BsaI | 0.5 | BsaI | 0.5 | BsaI | 0.5 |
12 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 |
13 | T4 Buffer | 1.5 | T4 Buffer | 1.5 | T4 Buffer | 1.5 |
14 | 10X BSA | 1.5 | 10X BSA | 1.5 | 10X BSA | 1.5 |
15 | H20 | 7.09 | H20 | 6.78 | H20 | 8.65 |
Assembly: cassettes for BioBrick characterization
Procedure - Making Competent Cells
1.
Added 100uL of seed culture to 900uL LB media (previously prepared) in a cuvette
2.
Blank the spectrophotometer with 1mL LB media
3.
Measured the OD600 of the culture (0.94)
4.
add 50ul of seed culture to 4.95mL of LB media for a total volume of 5mL
a.
The doubling time of DH5a cells is approximately 30 minutes. Incubate until the OD is about 0.2 (ok if a bit larger)
b.
Cells in incubator at 3:34 pm. Cells were taken out at 4:52 pm
c.
at pm, OD = 0.245
5.
Centrifuged at 4 degrees C for 10 min at 2500rpm
a.
observed cell pellet at the bottom of the tube
6.
Discarded supernatant
7.
Resuspended cell pellet in 1.6mL pre-chilled TSS buffer (pre-prepared)
8.
Distributed competent cell solution across (32) 1.5mL tubes to make 50uL aliquots
9.
Place into a box in -80 freezer
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Saturday, 9/16/17
People present Anna
Lab Silberg, Keck 201
A | B | C | |
1 | PgntK part plasmid | cys PUWA part plasmid | |
2 | DNA (ul for ~1 ug) | 5.60 | 16.20 |
3 | CutSmart 10X Buffer (ul) | 5 | 5 |
4 | EcoRI-HF (ul) | 2.5 | 2.5 |
5 | PstI-HF (ul) | 2.5 | 2.5 |
6 | H2O (ul) | 34.40 | 23.80 |
7 | Total | 50 | 50 |
Double Digest of sbp and chrR6 part plasmids
Week of 09/11
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-11 to 2017-09-17
Sunday, 9/17/17
People present Anna
Lab Silberg, Keck 201
Ran cys and PgntK digests on a gel; gel purified PgntK (bands for cys were indistinguishable since they are 3kb and 4 kb). Ligated PgntK into the BioBrick backbone and transformed into HS competent E. coli MG1655
Week of 9/18
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-20 to 2017-09-22
Wednesday, 9/20/17
People present: Albert, Anna
Time in lab: 8am-10am, 8pm-10pm
A | B | C | D | |
1 | ChrB | cys + PgntK | ||
2 | Reagent | Volume (uL) | Reagent | Volume (µL) |
3 | p439 | 1.6 | p439 (10x dil) | 1.6 |
4 | Con L4 (505) 10x | 1.9 | 501 (LS) | 0.25 |
5 | ChrB (10x) | 1.8 | PgntK | 0.29 |
6 | Con RE (520) 10x | 1.8 | cysPUWA | 2.76 |
7 | 530 | 0.22 | ||
8 | ||||
9 | BsaI | 0.5 | BsaI | 0.5 |
10 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 |
11 | T4 Buffer | 1.5 | T4 Buffer | 1.5 |
12 | 10X BSA | 1.5 | 10X BSA | 1.5 |
13 | H20 | 3.9 | H20 | 5.88 |
Cassette assembly: ChrB and cys
Comparing Growth of Shewanell and E. coli in wastewater
Obtained E. coli w/ F plasmid expressing RFP from Shelly.
Plated on Kan25 plate; in 37C incubator at 5:45 pm
Will take plate out at 9:45 am Thursday 9/21
Obtained Shewanella w/ chromasomally integrated GFP from Ilenne
Plated on LB only plate; in 30C incubator at 6:02 pm
Will take plate out at 10:02 am Thursday 9/21
PgntK BioBrick Liquid Culture
Made liquid culture of PgntK BioBrick in SOB + Chl34. Placed into the incubator at 9pm
Transformations cys and ChrB cassettes
Transformed cys and ChrB cassettes into HS competent E. coli MG1655. Plates were put into the incubator at 9:50pm.
Week of 9/18
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-20 to 2017-09-22
Thursday, 9/21/17
People present: Albert, Anna
Time in lab:
Goal: make liquid cultures for E. coli and Shewanella and take fluorescence measurements at several time points, prepare samples for sequencing, miniprep PgntK BioBrick, order primers
A | B | C | D | E | F | G | H | |
1 | Sample | ng/ul | volume of sample for 500 ng (ul) | volume of primers (ul) | volume of water (ul) | primer ID | name on order | # on order |
2 | ChrB3 | 113.0 | 4.5 | 2.5 | 8 | p15A | ChrB3 cass_fwd | 1 |
3 | ChrB3 | 113.0 | 4.5 | 2.5 | 8 | AmpR | ChrB3 cass_rev | 2 |
4 | lacI cassette | 224 | 2.3 | 2.5 | 10.2 | p15A | lacI cass_fwd | 3 |
5 | sbp 2 cassette | 627.1 | 0.8 | 2.5 | 11.7 | p15A | sbp2 cass_fwd | 4 |
6 | sbp 2 cassette | 627.1 | 0.8 | 2.5 | 11.7 | AmpR | sbp2 cass_rev | 5 |
Samples for Sequencing
PgntK miniprep
Followed Protocol 9
E. coli and Shewanella Liquid Culture
Picked one colony from E. coli plate and one colony from Shewanella plate into 4 mL LB.
Shewanella liquid culture had no antibiotic
E. coli liquid culture had 25 uL/mL Kan
E. coli culture went into 37C gyratory incubator at 4:45 pm.
Shewanella culture went into 30C gyratory incubator at 4:45 pm
Plain LB media culture also went in 37C incubator to determine if cultures were contaminated
Cultures will be taken out of incubator at 8:45 am Friday 9/22
Week of 9/18
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-20 to 2017-09-22
Friday, 9/22/17
Shewanella and E. coli cultures were taken out at 9:47 am.
LB control was clear: no contamination
Three glycerol stocks each of Shewanella and E. coli were prepared
500 uL of overnight culture and 500 uL of 50% glycerol were added to a 1.5 mL microcentrifuge tube
Week of 9/25
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-27 to 2017-10-02
Wednesday, 9/27/17
People present:
Time in lab:
Goal: Assemble Pcon cassete for BioBrick characterization, assemble nemA cassette (why not?) make liquid cultures from ChrR6 and KS
A | B | C | D | |
1 | Pcon | nemA | ||
2 | Reagent | Volume (uL) | Reagent | Volume(uL) |
3 | 439 | 0.2 | p439 | 0.2 |
4 | 501 (conLS) | 0.25 | p502 (ConL1) | 0.2 |
5 | Pcon(476) | 0.2 | Pchr (50fmol/ul, need 40 fmol) | 1.25 |
6 | RBS(457) | 0.26 | nemA (50fmol/ul, need 40 fmol) | 1.25 |
7 | 454 (mCherry) | 0.2 | p531 (OpconR2) | 0.2 |
8 | terminator (463) | 0.21 | ||
9 | conR1 (511) | 0.2 | ||
10 | ||||
11 | BsaI | 0.5 | BsaI | 0.5 |
12 | T4 DNA Ligase | 0.5 | T4 DNA Ligase | 0.5 |
13 | T4 Buffer | 1.5 | T4 Buffer | 1.5 |
14 | BSA + PEG --- in "E. coli toolkit" box | 1.5 | BSA + PEG --- in "E. coli toolkit" box | 1.5 |
15 | H20 | 9.48 | H20 | 7.9 |
Cassette Assembly
Catherine: I prepared the 2 GG assemblies and put them in the thermocycler with the red usb in it at 9:43am. I also pulled the KS and chrR6 plates from yesterday and wrapped them in the fridge. I will come back in the afternoon to start liquid cultures from the 2 plates and do transformations of the 2 assemblies.
Making competent cells
1.
Added 100uL of seed culture to 900uL LB media (previously prepared) in a cuvette
2.
Blank the spectrophotometer with 1mL LB media
3.
Measured the OD600 of the culture (0.94)
4.
add 50ul of seed culture to 4.95mL of LB media for a total volume of 5mL
a.
The doubling time of DH5a cells is approximately 30 minutes. Incubate until the OD is about 0.2 (ok if a bit larger)
5.
Centrifuged at 4 degrees C for 10 min at 2500rpm
a.
observed cell pellet at the bottom of the tube
6.
Discarded supernatant
7.
Resuspended cell pellet in 1.6mL pre-chilled TSS buffer (pre-prepared)
8.
Distributed competent cell solution across (32) 1.5mL tubes to make 50uL aliquots
9.
Place into a box in -80 freezer
A | B | C | D | E | F | G | H | I | |
1 | sample | concentration (ng/uL) | amount to use in reaction (of 10 x dilution) | Q5 Reaction Buffer | dNTPs | Fwd Primer (B17) | Rev Primer (B18) | Q5 DNA polymerase | H2O |
2 | ChrR6 c1 | 195.50 | 0.6 | 10 | 1 | 2 | 2 | 0.5 | 33.9 |
3 | p431 (+ control) | 84.5 | 1.3 | 10 | 1 | 2 | 2 | 0.5 | 33.2 |
4 | LacI3 | 128.50 | 0.6 | 10 | 1 | 2 | 2 | 0.5 | 33.9 |
5 | LacI4 | 96.90 | 1.3 | 10 | 1 | 2 | 2 | 0.5 | 33.2 |
Chr6 cassette 1 PCR
Week of 9/25
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-27 to 2017-10-02
Friday, 9/29/17
E. coli, Shewanella fluorescence standard curve:
1.
plated 1000x dilution of E. coli and Shewanella stock on Kan25 and plain LB plates, respectively
2.
E. coli in 37C incubator and Shewanella in 30C incubator at 4:56 pm
3.
Will take out at 9:00 am on 9/30
4.
Plan: pick 12 colonies each, culture in M9 in shallow-well plate; measure fluorescence overnight on TECAN
A | B | |
1 | Pcon | |
2 | Reagent | Volume (uL) |
3 | 438 | 0.25 |
4 | ConL4(505) | 0.2 |
5 | PglpT | 0.5 |
6 | BCD2 (456) | 0.2 |
7 | mCherry (454) | 0.2 |
8 | terminator (461) | 0.2 |
9 | ConR1 (511) | 0.2 |
10 | ||
11 | BsaI | 0.5 |
12 | T4 DNA Ligase | 0.5 |
13 | T4 Buffer | 1.5 |
14 | BSA + PEG | 1.5 |
15 | H2O | 9.25 |
Test assembly
Week of 9/25
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-27 to 2017-10-02
Saturday, 9/30/17
E. coli, Shewanella fluorescence standard curve
1.
plates were taken out at 10:54 am
2.
both were overgrown; will try plating again on Monday
Week of 9/25
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-27 to 2017-10-02
Sunday, 10/1/17
CD: Miniprepped the liquid cultures of cys cassette colonies. (There were 2 plates of cys cassete colonies that Shyam prepared; I took 2 colonies from 1 plate and 1 colony from the other to make a total of 3 liquid cultures. One of the liquid cultures gave a weird pellet upon centrifugation, so I discarded this one.) I then performed a test digest of the 2 cys cassette minipreps using p431 as a control (I didn't use p439 which is the backbone that the cassette was assembled into bc this plasmid does not have Esp3I sites.) The virtual test digestion results are below. Achieving these results on a gel would indicate proper assembly of the cys cassette which would be further verified by sequencing. I ran the test digestion samples on the gel as well as 1 ul of each of the miniprepped samples without any treatment as a secondary confirmation of plasmid size.
NanoDrop of Samples
cys Cassette A = 220.4 ng/ul
cys Cassette B = 136.2 ng/ul
A | B | C | D | |
1 | uL for cys cassette A dig | ul for cys cassette B dig | ul for 431 (+ control) dig | |
2 | water | 6.00 | 5.00 | 2.50 |
3 | 10X Tango Buffer | 1.00 | 1.00 | 1.00 |
4 | DNA (~500ng) | 2.50 | 3.50 | 6.00 |
5 | Esp3I | 0.50 | 0.50 | 0.50 |
6 | TOTAL | 10.00 | 10.00 | 10.00 |
Test digestion of cys cassette w/ Esp3I (incubate @ 37C for 1 hour)
Week of 9/25
Project: Lab Notebook
Authors: Catherine Dunaway
Dates: 2017-09-27 to 2017-10-02
Monday, 10/2/17
A | B | C | D | E | F | G | H | |
1 | Miniprep ID | ng/ul | vol for 500 ng (ul) | volume of primer (ul) | volume of water (ul) | primer ID | name of seq on order | #on order |
2 | cys cassette A | 220.4 | 2.3 | 2.5 | 10.2 | C54 | cys cassA_fwd | 1 |
3 | lacI 4 cassette | 96.9 | 5.2 | 2.5 | 7.3 | C54 | lacI cass_fwd | 2 |
4 | chrB 2 cassette | 111.3 | 4.5 | 2.5 | 8.0 | C54 | chrB cass_fwd | 3 |
5 | chrR6 cds BioBrick | 250 | 2.0 | 2.5 | 10.5 | VF2 | chrR6 BB_fwd | 4 |
6 | sbp cds BioBrick | 226.2 | 2.2 | 2.5 | 10.3 | VF2 | sbp BB_fwd | 5 |
7 | Pgntk+rbs BioBrick | 109.5 | 4.6 | 2.5 | 7.9 | VF2 | Pgntk BB_fwd | 6 |
8 | cys cassette A | 220.4 | 2.3 | 2.5 | 10.2 | C55 | cys cassA_rev | 7 |
9 | lacI 4 cassette | 96.9 | 5.2 | 2.5 | 7.3 | C55 | lacI cass_rev | 8 |
10 | chrB 2 cassette | 111.3 | 4.5 | 2.5 | 8.0 | C55 | chrB cass_rev | 9 |
11 | sbp cds BioBrick | 226.2 | 2.2 | 2.5 | 10.3 | VR | sbp BB_rev | 10 |
Sequencing Order
