Notebook | UIUC_Illinois

NOTE: LC21 - MC4100, LC22 - MC4100Z1, LC14 - MC4100, LC99 - MC4100Z1

May

6/30 Monday

First project meeting
Discussed potential project ideas

June

7/6 Monday

Narrowed potential project ideas to three:

Nitrogen Cycle
Degradation of Chemical UV Filters (Benzophenone-3)
Elastin

7/12 Monday

Decided to wok on the environmental track
Planning to meet with a professor for human practices
Summer camp outreach practices

7/26 Monday

Finalized on Biofilm as main topic of project

July

7/12 Tuesday

Started lambda red knockout prep
Created 200 mL of 10% glycerol stock

20 mL 100% glycerol + 180 mL DI water

Created 1L Tryptone Yeast Extract Agar (Himedialabs protocol)

6 g tryptone
3 g yeast extract
12 g agar

Created 1 SOB media
Cultured LC21, LC22, LC14, LC99 (LC24 Later) in LB-amp media for ~16 hours at 37 degrees C

LC21 = pKD 46 (time sensitive, don't grow above 30C)
LC22 = pKD3
LC14 = MC4100 strain of E. coli
LC99 = MC4100Z1 (tetracycline inducible)
LC24 = contains pCP20 (for flippase, don't grow above 30C)

7/13 Wednesday

Miniprep for LC21, LC22, LC14, LC99

Quiagen miniprep kit

7/14 Thursday

Plate	Yield	Purity
LC21	25.9	1.92
LC22	23.3	1.83
LC99	32.2	1.94
LC14	26.8	1.93
LC1172	43.9	1.90
LC24A (disposed)	32.1	1.83
LC24B (disposed)	50.4	1.82

Cultured LC24 in 30 degrees C for ~16 hours

7/18 Monday

Regrew overnight shake culture of LC24 in 30 degrees again because the miniprep data showed inconsistencies.
Transformation of pKD46 into MC4100 and MC4100Z1 (there was contamination in the shake culture, so we have to come again tomorrow to redo this transformation)

7/19 Tuesday

Did the miniprep on the LC24 from last time (Melissa)
Transformation of pKD46 into MC4100 and MC4100Z1 again, however we realized that pKD46 (LC21) had been grown wrong (we grew it in 37C instead of 30C), therefore we decided to redo the transformation again the next day.
Started an overnight shake culture of LC21 PCR on LC22- trial 1 (Vishnu)

Csga KO protocol followed

Gel electrophoresis- ran for est. 30 minutes

spotty gel
DNA at the very bottom of the gel- failed

7/20 Wednesday

LC22 (PKD3) PCR- trial 2- failed (Airah and Vishnu)

LC22 (PKD3) PCR- trial 2- failed (Airah and Vishnu)
diluted plasmid to 0.1 ng/ul- different than what we did on 7/19/2022
mixed dNTPs and rxn buffer- different from last time

LC22 (PKD3) PCR- trial 3- failed (Airah and Angela)

results: DNA running off the gel

Gel electrophoresis- ran for est. 30 mins

60 ml TAE buffer
0.6 g Agarose
0.6 ul stain

Gel Protocol:

Measure 0.6 g of Agarose and put it in a flask with 60 ml of TAE buffer

Microwave it for 45-50 seconds until Agarose is melted

Add the stain and swirl the mixture

Pour the mix into the gel holder and push bubbles to the side

Miniprep on LC21 (did two of them, labeled one A and the other B)

A: 59.5 ng/µL, 1.72 A260/A280
A: 68.0 ng/µL, 1.71 A260/A280
B: 41.7 ng/µL, 1.81 A260/A280
B: 37.1 ng/µL, 1.84 A260/A280

Redid transformation of pKD46 into MC4100 and MC4100Z1 again

Voltage 1800
Resistance 25
Capacitance 200
cuvette 1

7/21 Thursday

Innoculated LC 21 and LC 24 (LB + Amp)
Made LB media (Melissa and Airah)

7/22 Friday

Innoculated

LC 22- LB + Amp
LC 55- LB + Cm50
LC 1172- LB + Cm 50
LCpho 14- LB
LC 99- LB
Unlabeled?

Made stocks of LC 21 and LC 24

7/23 Saturday

Mini-prep performed on

LC21
LC22
LC24 (Twice)
LC1172 (Twice - A and B)
LC55

Stocks will be made of the following cultures:

LC1172 (twice)
LC55 (twice)
LCC22
LC14
LC99

7/26 Tuesday

Realized that transformations of pKD46 into MC4100 and MC4100Z1 was done on media that had no amp in them (the plates made were autoclaved after ampicillin was added to them), so (Melissa) started a new shake culture from the plated transformations from 7/20

7/27 Wednesday

Made new solid LB + amp (Vishnu and Melissa)
Plated the shake cultures from the day before( the transformations of pKD46 into MC4100 and MC4100Z) overnight

7/28 Thursday

Saw unsucessful growth of pKD46-transformed cells - we believe due to insufficient amount of cells plated

7/29 Friday

Regrew MC4100Z1 (LC99) and MC4100 (LC14) - Megan and Melissa

Grew in 25 mL SOB instead of 5 mL in 125 mL erlenmyer flask

Washing step with 25 mL 10% glycerol instead

Colonies picked of LC99 and LC14 from plate labeled 9/20 (has line dividing the two)

Transformation of pKD46 into MC4100Z1 and MC4100

followed "csgA KO" protocol in lab notebook
added 74.2 ng of plasmid (2 uL of 37.1 ng/uL plasmid from 7/20)
MC4100 (LC14) had to electroporate twice

Plated transformed MC4100Z1 and MC4100 (according to KO protocol)

pDK46 + MC4100Z1 on LB+amp
pKD46 + MC4100 on LB+amp
pDK46 + MC4100Z1 on LB (control)
pKD46 + MC4100 on LB (control)
grew 30 C overnight
plated 0.2 mL culture with the shaking beads

Autoclaved more water and made 400 mL of 10% glycerol stock

7/30 Saturday

Picked and innoculated plates #1 and #2 from 7/29 - Megan and Melissa

innoculated in 4.5 mL LB + amp100 (added 4.5 uL amp)
Plates showed lawn - a bit difficult to isolate single colony

7/31 Sunday

Created stocks of LC14 and LC99 according to "csgKO" protocol - Megan

750 uL cell culture + 750 uL 50% glycerol

LC14 stocks w/ 710 uL each, one of the LC99 stocks has a bit less than 750 uL

stored in -80 freezer by autoclave in box labeled "iGEM" (top right drawer/box shelf on top shelf)

August

8/1 Monday

PCR amplified pKD3 (LC22) twice - Vishnu

Possible mistakes in first one

Not mixed enough
Air bubbles (significant)
Old LC22 miniprep
Vortexed Tube before PCR

Ran Gel for LC22 PCR - Airah, Megan, Vishnu

Procedure

Add 5 microliters of Loading Dye into each PCR tube
Vortex the PCR tubes
Add 5 microliters of Plasmid (control), 1 microliter of Loading Dye for the Control
Add 5 micrliters of Loading Dye to each PCR sample
Used 5 uL ladder

Gel results:

8/2 Tuesday

Regrew MC4100 (LC14) + MC4100Z1 (LC99) for transformation (due to lawn plates) - Megan

25 mL culture
washing with 25 mL culture - 5 mins at 4000 rpm

Restreaked plates from 7/30 (due to lawn plates) - Megan and Angela

LB + amp - MC4100 w/ pKD24
LB + amp - MC4100Z1 w/ pKD24
LB - MC4100 w/ pKD24
LB - MC4100Z1 w/ pKD24

Transformed pKD46 into MC4100 and MC4100Z1 - Megan
Plated transformants (8 plates total):

100 uL LC14 on LB + amp
100 uL LC14 on LB
100 uL LC99 on LB + amp
100 uL LC99 on LB
200 uL LC14 on LB + amp
200 uL LC14 on LB
200 uL LC99 on LB + amp
200 uL LC99 on LB

Created stocks of LC14 and LC99

added 5 mL 50% glycerol into a 5 mL resuspension of LC14 and LC99 - step before transformation (in -80, in 50 mL tube)
added 0.4 mL of 50% glycerol into 0.4 mL transformed LC14 and LC99 (in -80)

PCR amplified pKD3 (LC22) @ 50 degrees Celsius Holding Temperature
Created 450 mL of LB agar, ~22 plates

8/3 Wednesday

Ran pKD3 (LC22) PCR amplicons on Gel

Bands were streaky and light, but still present

PCR amplified pKD3 @ 55 C and w/ 5 min final extension - Angela

8/4 Thursday

Ran gel of PCR pKD3 amplicons - Megan, Airah

Sample 1: Band @ 1 KB => used for cleanup

PCR cleanup of PCR pKD3 amplicons from 8/3 (Sample 1, lane 3) - Megan, Airah NEB Protocol:

eluted with 10 uL of elution buffer (9 left bc we checked w 1 uL)

Concentration: 21.4 ng/μL
Purity: 1.87

Redid pKD3 PCR - Airah

annealing temp at 55, final extension 5 mins (new protocol adjusted by Rao)
redid to gather more pKD3 plasmid - only one sample worked previously
35 cycles instead of 30
sample 1 and 4 have bright bands, 2 and 3 have faint bands

primer dimer!

add DMSO for next PCR?
or GC enhancer?

PCR cleanup on samples 1 & 4 - Airah

saved samples 2 and 3 if we need more for PCR cleanup
NEB protocol as #2
combined pKD3 amplicons "pKD3 amp"- 34 ng/uL with A260/A280 of 1.69

Grew MC4100 and MC4100Z1 in 125 mL SOB each (according to protocol) - Megan

added arabinose late -3 hours in vs 2 hours in

Centrifuged and suspended 125 mL cultured cells - Megan

split each 125 mL culture into 3 50 mL tubes

~40 mL of culture in each tube

washed with 40 mL of 10% glycerol per tube

Final resuspensions:

1 mL resuspensions in 10% glycerol of MC4100 (x 2)
2 mL resuspension in 10% glycerol of MC4100
1 mL resuspensions in 10% glycerol of MC4100Z1 (x 2)
2 mL resuspension in 10% glycerol of MC4100Z1
stored resuspensions in 4 degree fridge

Transformation of pKD3 - Megan

transformed 25 uL cells w/ 3 uL (75.3 ng) plasmid

pKD3 amplicon #1 was 25.1 ng/uL - used in transformation
transformation done 4 times

1 mL resuspension MC4100 (arced)
2 mL resuspension MC4100
1 mL resuspension MC4100Z1 (arced)
2 mL resuspension MC4100Z1 (arced, had to remake)

plated transformed cells into 8 plates total:

TYP + cmp - 1 mL resuspension MC4100 (0.9 mL)
TYP + cmp - 2 mL resuspension MC4100 (0.9 mL)
LB - 1 mL resuspension MC4100 (0.1 mL)
LB - 2 mL resuspension MC4100 (0.1 mL)
TYP + cmp - 1 mL resuspension MC4100Z1 (0.9 mL)
TYP + cmp - 2 mL resuspension MC4100Z1 (0.9 mL)
LB - 1 mL resuspension MC4100Z1 (0.1 mL)
LB - 2 mL resuspension MC4100Z1 (0.1 mL)
could not find TYG plates - should we make some?

8/5 Friday

Results from 8/4 transformation: control plates showed colonies but TYG+cmp did not

pKD3 PCR with 35 cycles- Airah
Gel electrophoresis- Airah

saved sample 4 (-20C) because other samples were faint
ran 35 mins total
bright primer dimers

for next time, we'll add DMSO so inhibit secondary structures from the primers

Regrew MC4100 and MC4100Z1 from single colony from plate - Megan

added 125 uL arabinose at 2 hr mark
cells took 4 hours 10 mins to reach OD 0.6

Centrifuged + resuspended 125 mL cultures - Megan and Airah

same as 8/4 protocol

Transformation with pKD3 - Megan

pKD3 plasmid 34.0 ng/uL, added 2.9 uL of plasmid with 25 uL cells - 98.6 ng DNA
used LB to revive cells after electroporation instead of SOC

prewarmed 1mL LB in 4 tubes for 4 transformations

8/6 Saturday

8/5 Transformation was unsuccessful

8/8 Monday

Redid PCR on pKD3 - Airah

used DMSO (1uL) to reduce primer dimers

Regrew MC4100 and MC4100Z1 for transformation - Megan

same protocol as previous days

Gel extraction of pKD3 amplicons

concentration: 18.4 ng/uL purity: 1.71

Centrifuged and resuspended MC4100and MC4100Z1 cultures

this time, collected entire culture into one 50 mL tube
added 8 mL glycerol per centrifuge step
final resuspension in 1 mL

Transformation of MC4100 and MC4100Z1

used pre-warmed LB again, see 8/5 for details
used amplicon from PCR cleanup (34.0 ng/uL, added 2.9 uL for 98.6 ng in transformation)

Plated transformants (4 plates total)

For both MC4100 and MC4100Z1:

50 uL culture onto control plate
950 uL culture onto TYG+cm25 plates

8/9 Tuesday

8/8 transformation results - failed

might be because of insufficient amplicon - we have been using the PCR cleanup sample

Transformation with cells grown 8/8

same protocol as 8/8
used gel extracted DNA vs. amplicon - 58.2 ng/uL plasmid DNA

3 uL of 19.4 ng/uL

PCR- pKD 3- same protocol as 8/5

used DMSO again (1uL)
to gather more amplicon (hopefully with higher concentrations)

Gel electrophoresis

correct band size: 1100
Voltage: 120
Time: 30 minutes

Gel extraction instead of PCR cleanup to get DNA only from amplicon

concentration very low - did not save amplicon

8/10 Wednesday

Grew MC4100 and MC4100Z1 in LB + amp (SOB previously)

125 uL amp100 into each 125 mL culture
grew to ~0.7 OD (previously 0.6)

Created Cm25 stock

10 mL 100% ethanol w/ 0.25g chloramphenicol
filtered with syringe filter (in cabinet on row with electroporation machine)

Created LB Agar (500 mL) + cm25 plates

added 500 uL cm25 into LB Agar mix
plated ~30 per plate - used thicker plates

PCR of pKD3 (x2)

4 50 uL samples
loaded 5 uL for gel, saved 45 uL for PCR cleanup
new primers came, ran another PCR with new primers (4 samples)

PCR cleanup of gel with new primers

Nanodrop conc + A260/A280

sample 1: 54.4 ng/uL, 1.71
sample 2: 37.0 ng/uL, 1.80
sample 3: 35.6 ng/uL, 1.83
sample 4: 39.8 ng/uL, 1.87

Gel electrophoresis with new primers

Gel electrophoresis with old primers

Centrifuged and resuspended MC4100 and MC4100Z1 cultures

centrifuged 10 mins at 4000 rpm (3-5 mins previously)
washed with 10 mL glycerol

Transformed MC4100 and MC4100Z1

used sample #4 from today - added 2 uL for 78.6 ng pKD3
used pre-warmed LB to save cells
plated on new LB + cm25 plates made 8/10

pre-warmed plates for an hour

50 uL on LB control plates, rest of culture on selective plates

8/11 Thursday

Transformation on MC4100 and MC4100Z1 failed

Grew MC4100 and MC4100Z1

grew 100 mL culture instead
split into 2 50 mL tubes + washed each tube with 40 mL 10% glycerol
resuspended each tube with 500 uL
made 3 aliquots of 50 uL for each strain

Transformed cells

mixed amplicon samples - pKD3 sample #3 w/ concentration of 41.1
added 2 uL = 82.2 ng pKD3 amplicon
mixed amplicon + cells in a 1.5 mL tube before adding to cuvette (previously added directly to cuvette)

Grew cells same as 8/10

8/23 Tuesday

Changes made to the chromosomal KO

add 100 uL of cells
10uL of DNA
1000 uL of LB
antibiotic Cm to a concentration of 20 (maybe)
don't vortex the cells with the glycerol washes
change electroporator settings to 0.2 cm but keep everything else the same
Check OD for 0.4 instead of 0.6
store cells in -80 by adding 10% glycerol
acKA KO

100 bp
Annealing Temp: 55 C

Dilute overnight cultures to 10 times (ex: 30 mL to 300 mL)
After the 37 degree shaker bring to 4 degree (cold) first, leave in ice for like 10 mins before centrifuging
Lower rpm maybe 4000 for 2 minutes
3 glycerol washes in all!
500 muL in last glyerol wash
Dilute to 100
Cells did not grow
Ran gel on ackA pKD3 amplicon

8/24 Wednesday

Made an overnight cultures of MC4100 and MC4100Z1

1 mL LB and 1uL amp

8/25 Thursday

Diluted o/n cultures according to revised protol
No cell growth
Made 3 o/n cultures to troubleshoot:

LC14 + 1 mL LB + 1 uL of Will's amp
LC99 +1 mL LB + 1 uL of Ebin's amp
LC14 + 1 mL LB + 0.5 uL of Ebin's amp

8/26 Friday

Saw growth in cultures a and c from last night
Diluted and grew o/n culture "a" according to revised protocol

grew to OD 0.44

Made cells electrocompetent according to revised protocol
Transformed LC14 - twice with csgA amplicon and twice with ackA amplicon

5 uL of amplicon (since we did not have enough for 10 uL each) + 100 uL cells

Made 5 stocks of 200uL each electrocompetent MC4100 + pKD46 cells
Made 6 plates -

LB - 50 uL ackA KO cells
LB - 50 uL csgA KO cells
LB + cm25 - rest of ackA KO cells (~950 uL) [x2, since 2 transformations]
LB + cs25 - rest of csgA KO cells (~950 uL) [x2, since 2 transformations]

8/29 Monday

made more LB Agar plates

12.5 g LB Miller broth
7.5 g Agar

8/30 Tuesday

Made more LB - looked cloudy and contaminated

25 g LB Miller

Made 1M L-arabinose solution

25 mL ddH20, 7.5g L-arabinose powder

Innoculated MC4100 and MC4100Z1 overnight in LB + amp (Revised csgA KO protocol)

8/31 Wednesday

Retried transformation using o/n colonies from 8/30

3 uL of amplicon per transformation - concentration 37.4

Ran pKD3 PCR to get more pKD3 amplicon

September

9/5 Monday

restreaked cell stock from 8/2 of LC14 + pKD46 and LC99 + pKD46 and grew o/n

9/6 Tuesday

grew cells from 9/5 restreak

50 mL cultures of each strain in LB + amp

made LB + cm plates
ran gel on amplicon from 8/31 - only sample 3 successful
PCR cleanup on sample 3

concentration: 45.7 ng/uL

tranformation of ackA amplicon and csgA amplicon - transformed LC14 in 1mm cuvettes and LC99 in 2 mm cuvettes; 4 transformations total

csgA KO in 1 mm cuvette
ackA KO in 1 mm cuvette
csgA KO in 2 mm cuvette
ackA KO in 2 mm cuvette

plated transformants (8 plates total, 4 control)

9/19 Monday

Made new LB media + autoclaved 125 mL flasks
Created amp100 stock

1 g in 10 mL water
1 mL aliquots - put in -20 freezer

9/21 Wednesday

grew LC99 and LC14 for pKD46 transformation

grew to OD ~0.5
glycerol steps 25, 15, 10, 5 mL

transformed 2 times

25 uL cells
4 uL plasmid, 26.6 ng/uL
LC14 arced, had to repeat x2

Plated 2 control, 2 experimental on LB+amp

0.2 mL on each plate

9/22 Thursday

successful transformation of LC14 and LC99

not much growth on LC14 plates

9/26 Monday

Grew an o/n culture of LC99 + pKD46 for pKD3 transformation and cell stock

grew later to ensure that o/n culture is taken out 16-20 hours later
3 mL of LC14
4 mL of LC99 (1 mL reserved for pKD3 transformation)

9/27 Tuesday

o/n cultures very clear

did not do pKD3 transformation, cells did not grow

October

10/5 Wednesday

Sucessful pKD3 transformants

10/7 Friday

colony PCR to verify the KO (25 uL samples)

5 uL NEB 5x Q5 reaction buffer
0.5 uL dNTPs
1.25 uL check primer 1 (fwd)
1.25 uL check primer 2 (rvs)
colony
0.25 uL Q5 polymerase
5 uL GC enhancer
11.75 uL nuclease free water
created master mix

Ran gel to verify band sizes

expect ~453 = size of csgA, ~1200 = size of ackA

Lanes:

DNA ladder
MC4100 colony - negative control
MC4100 csgA KO colony
MC4100Z1 csgA KO colony
MG1655 csgA KO colony
MC4100 ackA KO colony

VISHNU: PROMOTER CHARACTERIZATION
Measured GFP Flourescence

Prepare 12 Wells of 300 microliters for each of the 5 tubes

One at 10x dilution (30 microliters culture, 270 microliters water)
One at 20x dilution (15 microliters culture, 270 microliters water)
One of pure culture

Measure flourescence using Tecan Microplate Reader

Excitation Wavelength: 475 nm
Emission Wavelength: 508 nm
Make sure to reduce gain significantly

Results

Breakdown

Row 1 (B-> E, H-> L): Water Blanking
Rows 2-6: Flourescence from 0mM, 50mM, 100 mM, 150mM, and 200mM Sodium Nitrate, respectively

B -> E: 10x dilution
H-> L: 20x dilution

Plotted change in GFP flourescence at different concentraitions

GFP Flourescence decreased as Sodium Nitrate concentration increased (with some fluctuations)
rate of decrease DECREASEd as concentration increased

10/9 Sunday

VISHNU: PROMOTER CHARACTERIZATION
Overnight Culture Preparation w/ Different Colonies

Label and choose 5 colonies from transformed plate
Inoculate each colony in a separate tube with 5 mL LB + Kanamycin (1:1000 ratio)
Prepare 100 mM Sodium Nitrate Solution (accidentally used 1 M Sodium Nitrate due to Power of Ten error)
Add respective amounts of Sodium Nitrate to teach tube

- 0.26 mL for 5 mM tube
- 0.555 mL for 10 mM tube
- 0.88 mL for 15 mM tube
- 1.25 mL for 20 mM tube

Grow overnight in incubator (12-16 hrs, 30 C, 250 rpm)

10/10 Monday

VISHNU: PROMOTER CHARACTERIZATION
Overnight Culture Preparation w/ Same colony

Add approximately 450 microliters from the overnight culture tube with NO Sodium Nitrate to 5 separate tubes
Add 4.5 mL LB + Kanamycin to each of the 5 tubes
Prepared 100 mM Sodium Nitrate Solution (after realizing my Power of Ten Error)
Add respective amounts of Sodium Nitrate to teach tube

- 0.26 mL for 5 mM tube
- 0.555 mL for 10 mM tube
- 0.88 mL for 15 mM tube
- 1.25 mL for 20 mM tube

Grow overnight in incubator (12-16 hrs, 30 C, 250 rpm)

10/11 Tuesday

VISHNU: PROMOTER CHARACTERIZATION
Measured GFP Flourescence

Prepare 8 Wells of 300 microliters for each of the 5 tubes

One at 10x dilution (30 microliters culture, 270 microliters water)
One at 20x dilution (15 microliters culture, 270 microliters water)

Measure flourescence using Tecan Microplate Reader

Excitation Wavelength: 475 nm
Emission Wavelength: 508 nm
Make sure to reduce gain significantly

Results

GFP Flourescence decreased with increasing concentrations

Saw an increased from 5mM to 10mM Nitrate, however

Rate of decreased DECREASED from 5 to 10, then started increasing again

May