Lab Notebook

General Anoxic HGT Biosensor Design

May

Week 1

Week 2

05/26 - 06/01

Wednesday

Completed site specific training
Grew 5 mL E. coli st. WM3064 pRL814-GFP in LB, spectinomycin, and DAP
Shook at 37 ℃ overnight
Streaked a spec+LB+agar plate with WM3064

Thursday

Made a cryostock of WM3064 pRL814-GFP
Used the OMEGA bio-tek E.Z.N.A plasmid miniprep kit I to purify the pRL814 plasmid from the culture tubes
Checked concentrations of purified plasmid
Prepared 100mM primer stock solutions of pRL814-F-JE and pRL814-R-JE
Ran PCR to amplify the pRL814 backbone with primers
Ran a DNA gel for the two PCR samples:

Did PCR cleanup of pRL814-GFP and checked the final concentrations

Friday

DPNI digest of the pRL814-GFP samples
Made a sleeve of LB + agar +spectinomycin plates
Gibson assembly with two reactions

Gibson Assembly (1)

Additive	Amount (uL)
Insert	4 uL
pRL814-GFP backbone	1 uL
2x high-fidelity master mix	10 uL
dd H₂O	20 uL

Gibson Assembly (2)

Additive	Amount (uL)
Insert	1 uL
pRL814-GFP backbone	1 uL
2x high-fidelity master mix	2 uL

Transformed both Gibson assemblies and plated on LB + agar + DAP + spectinomycin plates

June

Week 3

06/02 - 06/08

Monday

No colonies on plates from 5/31
Redid transformation of Gibson assemblies 1 and 2
Did another Gibson Assembly (3)

Gibson Sample (3)

Additive	Amount (uL)
Insert	3 uL
pRL814-GFP backbone	3 uL
2x high-fidelity master mix	6 uL

Negative Control

Additive	Amount (uL)
dd H₂O	3 uL
pRL814-GFP backbone	3 uL
2x high-fidelity master mix	6 uL

Transformed 2 uL of Gibson Assembly (3) and negative control, each into 50 uL of competent WM3064

Tuesday

Checked plates from yesterday's transformation and saw no colonies
Repeated transformation of Gibson (3) using 10 uL of the gene into 100 uL of competent cells

Plated on LB + agar + DAP + Sp

Did Q5 PCR to amplify the pRL814-GFP backbone

Sample 1 annealed at 54 ºC
Sample 2 annealed at 58 ºC

Thursday

Checked the plates from the 6/4 transformation and there were colonies
Screened these plates with colony PCR
Ran DNA gel:

Order (left to right): Ladder, positive control (first four wells broke upon comb removal so the 3 bands to the right of the ladder are all pRL814-GFP positive control), samples 10-2, last two wells both contain sample 1 (broke and leaked into each other)
Selected four samples with the cleanest bands to grow cultures of 1,2,9, and 10

Friday

Set up a plate reader with a 96-well, black, flat-bottom plate
Made a cryostock of each of the four cultures and stored at -80 ºC
Purified plasmid from each culture using the Omega Mini Kit I and checked concentrations

Sample 1 = 70.9 ng/uL
Sample 2 = 78.0 ng/uL
Sample 3 = 123.9 ng/uL
Sample 2 = 70.8 ng/uL

Week 4

06/09 - 06/15

Monday

Compiled and interpreted plate reader data using the programs R and Rstudio
Prepared and submitted eight samples for Sanger Sequencing
Inoculated pRL814-fdhF-GFP samples 1,2,9,10 from cryostocks and a pRL814-GFP control in 10 mL culture tubes
Plated pRL814-fdhF-GFP samples 1,2,9,10 from cryostocks
Ran Q5 PCR to amplify the fhlA gene from the E. coliE. coli > WM3064 genome
- Negative control with water instead of DNA

Tuesday

Prepared a 96-well plate of pRL814-fdhF-GFP with variable formate concentrations

Formate concentrations of : 0, 25, 50, 75, and 100 mM

Negative control: pRL814-GFP with same concentrations
Positive control: pRL814-GFP induced with IPTG
Q5 PCR amplified pRL814-fdhF-GFP backbone

Negative control with water instead of DNA

Ran a DNA gel with the amplified backbone and fhlA fragment PCR products

Plasmid purified the remaining 3 mL from the liquid pRL814-fdhF-GFP cultures

Wednesday

Isolated genomic DNA from a 4 mL WM3064 culture
Repeated the PCR amplification of fhlA from the isolated genomic E. coli DNA
Ran the fhlA PCR products through electrophoresis

Left → right: ladder, negative control, (1), (2)
Repeated Q5 pcr amplification of the pRL814-fdhF-GFP backbone with a temperature gradient (51, 54, 57, and 60 °C)
Analyzed Sanger results of pRL814-fdhF-GFP and realized the forward primer used for the last submission was inadequate so samples were resubmitted with pCBB FWD 1

Thursday

DPNI digest of fhlA
Purified plasmid from each culture using the Omega Mini Kit I and checked concentrations
PCR cleanup of pRL814-fdhF-GFP and fhlA
Gibson assembly of fhlA into pRL814-fdhF-GFP
Transformed assembly in WM3064 and plated the transformation

Friday

Checked plates from yesterday’s transformation and saw no colonies
PCR cleanup the remaining undigested fhlA PCR product
Gibson assembly redone, transformed (10uL gene into 100uL competent cells), and plated
Also transformed negative gibson assembly (no insert added)

Week 5

06/16 - 06/22

Monday

Made cryostocks of WM3064 pRL814-fhlA-fdhF-GFP samples 1, 2, 3, and 4
Made cryostock of S. Oneidensis MR-1 wild type
Purified pRL814-fhlA-fdhF-GFP from samples 1, 2, 3, and 4
Began conjugation of pRL814-fhlA-fdhF-GFP from E. coli WM3064 to S. oneidensis MR-1.

Tuesday

SmaI digested pRL814-fhlA-fdhF-GFP (3) as well as pRL814-fdhF-GFP (10) for comparison
DNA gel with DPNI digests

HindIII digested the pRL814-fhlA-fdhF-GFP samples 1-4 and pRL814-GFP for comparison, as the SmaI digest was inconclusive
DNA gel with HindIII digests

Inoculated 4 colonies from S. oneidensis MR-1 pRL814-fhlA-fdhF-GFP plates, samples 1-4 of WM3064 pRL814-fhlA-fdhF-GFP from cryostocks, and pRL814-GFP from cryostock in 10 mL culture tubes

Wednesday

Prepared 96-well plate reader with four MR-1 and 4 WM3064 pRL814-fhlA-fdhF-GFP samples

Formate concentrations: 0, 15, 20, 30, 50, and 100 mM
Negative control: pRL814-GFP with same formate gradient
Control: pRL814-GFP induced with IPTG

Started 48-hr plate reader data collection)

Thursday

Analyzed sequencing results for pRL814-fhlA-fdhF-GFP

Sequences did not align with the template in the fhlA region
Results aligned with previous construct, lacking the addition of the fhlA genes

Sent the fhlA insert for Sanger sequencing
PCR amplification of pRL814-fdhF-GFP backbone (from our previously amplified and purified -20 °C stock of pRL814-fdhF-GFP)

Annealed at 55 °C
Extension time = 3.5 minutes

DNA gel of amplified PCR products

Friday

Checked plates from yesterday’s transformation and saw no colonies
PCR cleanup the remaining undigested fhlA PCR product
Gibson assembly redone, transformed (10uL gene into 100uL competent cells), and plated
Also transformed negative gibson assembly (no insert added)

Week 6

06/23 - 06/29

Monday

Made cryostocks of WM3064 pRL814-fhlA-fdhF-GFP samples 1, 2, 3, and 4

Included negative control with no DNA
Annealed at 53.5 °C
Extension time = 3.5 minutes

PCR amplified fhlA genes (from cleaned up -20 °C fhlA pcr product)
DNA gel; left to right: ladder, linear pRL814-fdhF-GFP backbone, negative control, fhlA

Ran larger DNA gel with the remaining pRL814-fdhF-GFP backbone and did a gel extraction and clean up.
Final concentration: 15.7 ng/uL - very poor peak, lots of background from the running dye

Tuesday

PCR amplified more pRL814-fdhF-GFP again, this time from the -20 °C stock of the already linearized backbone (doubled pcr reaction volume to 50 uL)
No band on the gel

HindIII digested the pRL814-fhlA-fdhF-GFP samples 1-4 and pRL814-GFP for comparison, as the SmaI digest was inconclusive
DNA gel with HindIII digests

Inoculated 4 colonies from S. oneidensis MR-1 pRL814-fhlA-fdhF-GFP plates, samples 1-4 of WM3064 pRL814-fhlA-fdhF-GFP from cryostocks, and pRL814-GFP from cryostock in 10 mL culture tubes

Thursday

Performed Gibson assembly with cleaned-up pcr products

4 uL fhlA insert
3 uL backbone
10 uL high-fidelity master mix
3 uL sterile water (to 20 uL total reaction volume)

Transformed 10 uL of each Gibson assembly product in ~100 uL E. coli WM3064
Made and autoclaved 200 mL LB and 80 mL SOC

Friday

Checked plates from yesterday’s transformation and saw no colonies
PCR cleanup the remaining undigested fhlA PCR product
Gibson assembly redone, transformed (10uL gene into 100uL competent cells), and plated
Also transformed negative gibson assembly (no insert added)

July

Week 7

06/30 - 07/06

Monday

Checked plates from Friday’s transformation and counted colonies

Negative: 24
Rxn 1: 16
Rxn 2: 7
Concentrated rxn 1: 19

Colony PCR

Tuesday

Checked plates from yesterday’s transformation:

No colonies on either assembly plate
Small colonies on negative control, scattered; localized to a small (quarter-sized) section of the plate

Q5 PCR amplified pRL814-fdhF-GFP (10)

Also amplified the unmodified pRL814-GFP for comparison
50 uL total reaction volume; used 4 uL DNA
Annealed at 55 ºC
Extension time = 3 minutes

DNA gel

Ladder, pRL814-fdhF-GFP backbone, original pRL814, fhlA

Results: bands for both backbones showed just over 3 kb, with faint bands also around 10 kb (both incorrect sizes). The band for fhlA is at the correct size of around 2.5 kb.

Q5 PCR amplified pRL814-fdhF-GFP (10) and unmodified pRL814-GFP again

50 uL reaction volume containing 4 uL DNA
Annealed at 53.5 ºC
Extension time = 3 minutes

Wednesday

DNA gel:

Ladder, pRL814-fdhF-GFP backbone, pRL814-GFP backbone (for comparison)

Checked plates from Monday’s transformation:

5 colonies had grown on the concentrated assembly plate!

Colony PCR

‘Colonies 1-3’ picked from and labeled on the concentrated plate
‘Colony 4’ picked from and labeled on negative control plate
Included pRL814-fdhF-GFP for comparison
Colony PCR gel (ladder, colonies 1, 2, 3, and 4, pRL814-fdhF-GFP)

Week 8

07/07 - 07/13

Monday

DNA gel of products from last Wednesday’s colony PCR

Ladder, colonies 3, 5, and 6, pRL814-fdhF-GFP

Realized we had two primers, both named pRL814-REV, and that previous colony PCRs had used the incorrect primer.
Redid colony PCR from last week with colonies 1-6

Included negative, pRL814-fdhF-GFP, and pRL814-GFP controls
Annealed at 61 ºC
Extension time = 2.5 minutes
Colony PCR gel (ladder, pRL814-fdhF-GFP, pRL814-GFP, colonies 1-6, negative control)

Forgot to include ladder…

Redid colony pcr with colonies 1 and 3, pRL814-fdhF-GFP, and pRL814-GFP

Annealed at 61 ºC
Extension time = 2.5 minutes

Tuesday

Ran DNA gel of colony PCR products

Ladder, pRL814-fdhF-GFP, pRL814-GFP, colony 1, colony 3

Results: Bands for both pRL814 controls appear at 1.5 kb, which is the correct size. Bands for colonies 1 and 3 appear around 500 base pairs. This indicates that the backbone was amplified to no longer contain lacI (~1 kb) and it presumably must have recircularized once the lacI was removed.

Wednesday

Checked Sanger sequencing results for fhlA pcr product

Aligned with pRL814-fhlA-fdhF-GFP construct on Benchling.
Sequences align very well along fhlA segment

PCR cleanup of pRL814-fdhF-GFP backbone

Final concentration = 57.6 ng/uL

DPNI digested cleaned-up pRL814-fdhF-GFP backbone

Incubated at 37 ºC for 1 hour instead of the protocol-recommended incubation time of 15 minutes
Final concentration calculated to be about 20 ng/uL

Gibson assembly

8 uL insert (~0.103 pmol)
4 uL clean, DPNI digested backbone (~0.01 pmol)
12 uL 2X high-fidelity master mix
Ran for 1 hour; included negative control with water instead of insert

Transformation of assembly and negative control

Transformed 10 uL of gene into ~100 uL chemically competent WM3064
Plates (one of each for assembly and negative control)

LB, spec., DAP - plated 20 uL of transformation
LB, spec., DAP, 5 mM Na+HCOO- - plated 20 uL of transformation
LB, spec., DAP - plated 50 uL of concentrated transformation (spun down cells and resuspended in 50 uL LB)
LB, spec., DAP, 5 mM Na+HCOO- - plated 50 uL of concentrated transformation (spun down cells and resuspended in 50 uL LB)

Thursday

Counted colonies from transformation:

No formate plate = 0/li>
Formate plate = 0
Concentrated no formate plate = 6
Concentrated formate plate = 2
Negative control plate = 0

Colony PCR of colonies 1-5 from concentrated no formate plate

Included pRL814 and pRL814-fdhF controls

DNA gel (pRL814-fdhF, pRL814, colonies 1-5, ladder)

Results: the ladder did not show up well, presumably due to pipetting of air bubbles into the well when loading the gel. Even so, colonies 1-4 are notably smaller than the controls (indicating possible recircularization without the fhlA insert during Gibson assembly). Colony 5 however, appears to be about 1 kb larger than the pRL controls. This is a promising indication that colony 5 contains the fhlA insert.

Grew overnight culture of colony 5 in 5 mL LB, sp., + DAP

Incubated at 37 ºC for approx. 20 hours. Stored cultures at 4 ºC

Repeated colony PCR with colony 5, colonies 6-12 from the concentrated no formate plate, colonies 13-14 from the concentrated formate plate, pRL814 and pRL814-fdhF controls.

Friday

DNA gel of colony PCR products:

Ladder, pfdhF, pRL814, colonies 5-14

Results:

Colony 5: correct size for pfhlA construct (~8 kb)!
Colonies 6-12: no bands…
Colonies 13 and 14: smaller bands than control, indicative of recircularization before fhlA was inserted

Colony count repeated:

No formate plate = 2
Formate plate = 0
Concentrated no formate plate = 27
Concentrated formate plate = 4
Negative control = 0

Week 9

07/14 - 07/20

Monday

Made 15 mL of 500 mM sodium formate and filter sterilized it.
Streaked overnight culture of WM3064 pfhlA (from colony 5) on 2 LB, sp., DAP plates

One plate had no sodium formate
One plate had 5 mM sodium formate

Plasmid prepared overnight culture of pfhlA

Final concentration = 120.5 ng/uL (yielded 40 uL = 4.82 ug)

Set pfhlA for Sanger sequencing

‘JE F’ = 9 uL plasmid, 1 uL pfhlA-FWD, 2 uL dH2O
‘JE R’ = 9 uL plasmid, 1 uL pRL814-REV, 2 uL dH2O

Inoculated MR-1 W.T. cryostock in 5 mL LB

Incubated at 30 ºC overnight

Inoculated WM3064 pRL814-GFP cryostock and WM3064 pfhlA (colony 5) in 5 mL LB, sp., DAP each

Incubated at 37 ºC overnight

Tuesday

Began conjugation of E. coli WM3064 pfhlA to S. oneidensis MR-1 W.T.

Incubated at 30 ºC on DAP plate for 8 hours
Streaked fresh sp. plate and incubated at 30 ºC overnight

Ran plate reader

Wednesday

Stopped plate reader at 20.25 hours, after growth curves indicated that the stationary phase had been reached.
Analyzed plate reader data in R Studio
Ran another plate reader experiment

Thursday

Made 400 mL LB + sp.
Grew overnight 5 mL cultures:

MR-1 pfhlA in LB + sp.
MR-1 pfhlA in LB + sp., 25 mM sodium formate
WM3064 pfhlA in LB + sp.
WM3064 pfhlA in LB + sp., 25 mM sodium formate
WM3064 pRL814 in LB + sp.
WM3064 pRL814 in LB + sp., 25 mM sodium formate

MR-1 was incubated at 30 ºC
WM3064 was incubated at 37 ºC

Friday

Plasmid prep’d pfhlA from 4 overnight cultures of MR-1 and 2 overnight cultures of WM3064.

Final concentration = 272.73 ng/uL
Final yield ~ 43.64 ug pure plasmid

Attempted to run endpoint fluorescence assay in plate reader, however the aerobic plate reader was in use and the anaerobic plate reader was not able to be programmed to the 475/509 excitation/emission of GFP.

Week 10

August

Week 11

08/04 - 08/10

Tuesday

Made 1 L M5

After autoclaving, added Wolffe’s nutrients, vitamins, and spec.
Added 20 mM D-Lactase for carbon source

Made 1 L M9

After autoclaving, added Wolffe’s nutrients, vitamins, 2 mL 1M MgSO4, 100 uL 1M CaCl2, and spec.
Added 0.2% glucose for carbon source

Made LB, spec. Plates

Wednesday

Plated MR-1 pRL814-GFP from cryostock on an LB, spec. plate
Plated WM3064 pRL814-GFP and pfhlA from cryostocks of LB, spec., DAP plates

Incubated all plates at room temperature for ~36 hours

Thursday

Met with Dr. Michaela TerAvest to discuss progress on the biosensor project
Lab closed for the day for cleaning and maintenance
Analyzed burden assay data in Rstudio using a script made by the UT Austin iGEM team

Friday

Streaked MR-1 pfhlA colonies from 7/16/19 conjugation plate onto a fresh LB, sp. plate
Site directed mutagenesis with the New England Biolabs Q5 Kit

25 uL reaction volumes
Annealing temperature = 62 ºC
Extension time = 3 minutes 50 seconds

Week 12

08/11 - 08/17

Sunday

Ran DNA gel with site directed mutagenesis pcr products, order: ladder, circular pure pfhlA, TTACAA rxn, CTACAA rxn
Started overnight liquid cultures of MR-1 and WM3064 in LB

MR-1 pfhlA 0 mM + 25 mM formate
MR-1 pRL814 0 mM + 25 mM formate
WM3064 pfhlA 0 mM + 25 mM formate
WM3064 pRL814 0 mM + 25 mM formate

Monday

Performed KLD reaction on site directed mutagenesis pcr products
Transformed 5 uL of each KLD reaction into 50 uL of competent E. coli WM3064
Took endpoint OD and GFP readings of overnight LB cultures in anaerobic plate reader

Tuesday

Colony count from transformations:

CTACAA = 37
TTACAA = 7

Inoculated transformants in 5 mL LB, sp., DAP. Incubated at 37 ºC overnight.
Normalized ODs of MR-1 LB overnights from Sunday to 1.0 and washed with M5 minimal medium. Inoculated MR-1 pRL814 and pfhlA in 5 mL M5 minimal medium without formate and M5 with 25 mM formate. Incubated and shook at 30 ºC overnight.

Wednesday

Took endpoint OD and GFP readings of overnight M5 cultures in anaerobic plate reader
Made cryostocks of MR-1 pRl814 and pfhlA from overnight LB cultures (JE 005 and 006). Also made cryostocks of WM3064 pfhlA mutants TTACAA and CTACAA
Performed plasmid prep of site directed mutagenesis transformants
Concentrations (ng/uL):

1 = 192.6
2 = 142.5
3 = 172.6 (jagged absorbance peak)
4 = 168.5
5 = 162.4 (jagged absorbance peak)
6 = 114.4 (very sloppy peak)
7 = 170.2
8 = 162.3

Prepared samples 1-8 for sequencing and stored at -20 ºC.
Grew LB overnights of WM3064 and MR1 pRL814 and pfhlA
Grew triplicate M5 overnights of MR-1 pRL814 and pfhlA each with and without 25 mM formate.

Friday

Took endpoint OD and GFP readings of 46-hour overnights
Submitted pfhlA mutants 1-8 for Sanger Sequencing
Made 400 mL of M5

Week 13

Week 14

September

Week 15

Week 16

Week 17

Week 18

Team:MichiganState/Notebook/Biosensor

Lab Notebook

May

05/20 - 05/25

Monday

Wednesday

Thursday

Friday

05/26 - 06/01

Wednesday

Thursday

Friday

Gibson Assembly (1)

Gibson Assembly (2)

June

06/02 - 06/08

Monday

Gibson Sample (3)

Negative Control

Tuesday

Thursday

Friday

06/09 - 06/15

Monday

Tuesday

Wednesday

Thursday

Friday

06/16 - 06/22

Monday

Tuesday

Wednesday

Thursday

Friday

06/23 - 06/29

Monday

Tuesday

Thursday

Friday

July

06/30 - 07/06

Monday

Tuesday

Wednesday

07/07 - 07/13

Monday

Tuesday

Wednesday

Thursday

Friday

07/14 - 07/20

Monday

Tuesday

Wednesday

Thursday

Friday

07/21 - 07/27

Monday

Tuesday

August

08/04 - 08/10

Tuesday

Wednesday

Thursday

Friday

08/11 - 08/17

Sunday

Monday

Tuesday

Wednesday

Friday

08/18 - 08/24

Sunday

Monday

Wednesday

08/25 - 08/31

Monday

Tuesday

September

09/01 - 09/07