iGEM 2022 — Team uiuc-illinois

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![Logo](https://static.igem.wiki/teams/4436/wiki/notebook.png)
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* [May](#May)
* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)
* [October](#October)

**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

![LC99 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2038.jpg)

![LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2040.jpg)

![LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2042.jpg)

![LC99 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2039.jpg)

### 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)

![LC99 and 14 Stocks](https://static.igem.wiki/teams/4436/wiki/lc99-and-14-stocks.jpg)

## 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

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

* Gel results:

![Gel Results](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-1.jpg)

### 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

![Black](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-black.jpg)

![White](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-white-1.jpg)

![Black 2](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-2-black.jpg)

![White 2](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-white.jpg)

+ 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

![pKD3-PCR-8_4](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-4-gel.png)

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

![PCR Cleanup Protocol](https://static.igem.wiki/teams/4436/wiki/pcr-cleanup.jpg)

+ 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

![35 Cycles](https://static.igem.wiki/teams/4436/wiki/pkd3-8-4-trial-2-35x-cycles.png)

- 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

![Transformation Results](https://static.igem.wiki/teams/4436/wiki/screen-shot-2022-10-09-at-8-20-07-pm.png)

* 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

![pKD3 PCR](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-5-1.png)

* 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

![8/5 Transformation](https://static.igem.wiki/teams/4436/wiki/8-5-transformation.jpg)

### 8/8 Monday

* Redid PCR on pKD3 - Airah

+ used DMSO (1uL) to reduce primer dimers

![pkD3 with DMSO](https://static.igem.wiki/teams/4436/wiki/pkd3-with-dmso-8-8.png)

* 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](https://static.igem.wiki/teams/4436/wiki/8-8-transformation.jpg)

* 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)

![PCR - pKD3 with DMSO 8/9](https://static.igem.wiki/teams/4436/wiki/pkd3-with-dmso-8-8.png)

* 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

![pKD3 PCR with new primers](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-w-new-primers-8-10.png)

* Gel electrophoresis with old primers

![pKD3 PCR 8/10](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-10.png)

* 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

![LB + cm25 LC14](https://static.igem.wiki/teams/4436/wiki/lb-cm25-lc14.jpeg)

![LB + cm25 LC99](https://static.igem.wiki/teams/4436/wiki/lb-cm25-lc99.jpeg)

![LB LC14](https://static.igem.wiki/teams/4436/wiki/lb-lc14.jpeg)

![LB LC99](https://static.igem.wiki/teams/4436/wiki/lb-lc99.jpeg)

* 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

![Transformation of LC14 and LC99](https://static.igem.wiki/teams/4436/wiki/transformaiton-of-lc14-and-lc99.jpg)

![LB LC99 + pkD46 and LC LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/lb-lc99-pkd46-and-lc-lc14-pkd46.jpg)

### 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

![LC99 and LC14](https://static.igem.wiki/teams/4436/wiki/68599107637-b09758b9-89bd-417a-a258-eedb145d68ad.jpg)

* did not do pKD3 transformation, cells did not grow

## October

---

### 10/5 Wednesday

* Sucessful pKD3 transformants

![Transformations](https://static.igem.wiki/teams/4436/wiki/img-1644.jpg)

### 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

![Colony PCR](https://static.igem.wiki/teams/4436/wiki/10-5-colony-pcr-2.png)

* 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

![Fluorescence](https://static.igem.wiki/teams/4436/wiki/image.png)

### 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

![More Fluorescence](https://static.igem.wiki/teams/4436/wiki/image2.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block headercontent %}
![Logo](https://static.igem.wiki/teams/4436/wiki/engineering.png)
{% endblock %}
{% block page\_content %}

## Engineering Design Cycle

---

We underwent the Engineering Design Cycle as we designed and tested our primer for csgA knockouts.

The pages for our Primers have been finished ( [http://parts.igem.org/Part:BBa\_K4436017](http://parts.igem.org/Part%3ABBa_K4436017) and  [http://parts.igem.org/Part:BBa\_K4436666](http://parts.igem.org/Part%3ABBa_K4436666) ) but the cycle was heavily followed in ensuring our success.

#### Design

Our primer design consisted of the following:

1. Finding 40 nucleotides of homology to the flanking region of the csgA gene (the one we’re trying to knockout)

2. Finding 20 nucleotides of homology to the flanking regions of the FLP recombinase sites in pKD3 plasmid

3. Combing them respectively to make the primer

#### Build

The primers were built by IDT as part of our collaboration with the company.

#### Test

We test our primers by conducting the PCR of pKD3 and running it on a gel to see if the amplification worked. More information can be found on our parts pages.

#### Learn

The first time PCR failed, we found homology regions lower in GC content and closer to the sites because we believed that’s why we failed. We then designed a second set of primers.

The second time it failed, we took a closer look and realized we placed the homology regions in the wrong order for the Reverse Primer. We then designed our third set of primers.

When that still wasn’t working, we lowered the annealing temperature and finally got great results, which can be found on our parts registry.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block headercontent %}
![Logo](https://static.igem.wiki/teams/4436/wiki/experiment.png)
{% endblock %}
{% block page\_content %}

## csgA KO (“csgA KO” and “Revised csgA knockout” protocols)

---

The first step in our experiments was to perform a csgA knockout (KO) using the lambda red method outlined by Datsenko and Wanner [1]. The purpose of the csgA KO is to replace the native csgA gene with our modified csgA-SpyTag plasmid construct.

**csgA KO Protocol:**  <https://drive.google.com/file/d/1P-U4E-h5uwOKAzR4gf0AbFunBmjLYv-z/view?usp=sharing>

**Revised csgA knockout Protocol:**  <https://drive.google.com/file/d/10Zagbu3OgQ0deAOk_LXAEj10ORWVhXyk/view?usp=sharing>

## Transformation of SpyTag/SpyCatcher Plasmids

---

We planned to co-transform csgA-SpyTag-GFP and SpyCatcher-nrtA-His into ΔcsgA mutants. We created 5 different variations of csgA-SpyTag and SpyCatcher-nrtA plasmids to test. Plasmid components are based off of existing iGEM biobrick parts designed by Marburg 2015.

![Plasmid Description](https://static.igem.wiki/teams/4436/wiki/experiments/screen-shot-2022-10-11-at-7-26-23-pm.png)

Plasmids 1, 3, and 4 act as controls to verify the correct expression of csgA-SpyTag, SpyCatcher, and SpyCatcher-nrtA. CsgA production in plasmids 1 and 2 would have been verified with Congo Red (CR) dye, which stains amyloid fibers. Successful transformations of plasmids 1, 2, and 3 would have been verified through detecting GFP fluorescence in transformed colonies. The His tag in plasmids 3, 4, and 5 would have been used in a Western blot to verify that the correct construct was produced. A Western blot would also have been used to verify the binding of SpyTag with SpyCatcher, since we know the size of the entire csgA-SpyTag-GFP-SpyCatcher-nrtA-His construct.

## Characterizing the Nitrate-Sensitive Promoter

---

The narG CC(-40.5) promoter was characterized by transforming Plasmid 2 into E. Coli DH5-A using the protocol outlined by New England Biolabs [2], then growing overnight cultures and using a microplate reader as outlined in the protocol we devised. Our goal was to determine the impact of varying concentrations on protein production, which was directly proportional to the amount of fluorescence we measured.

**References**

[1] K. A. Datsenko and B. L. Wanner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products,” Proceedings of the National Academy of Sciences, vol. 97, no. 12, pp. 6640–6645, Jun. 2000, doi: 10.1073/pnas.120163297.

[2]“High Efficiency Transformation Protocol (C2987H/C2987I).” NEB, https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987.

[3] Hothersall, J., Lai, S., Zhang, N., Godfrey, R. E., Ruanto, P., Bischoff, S., Robinson, C., Overton, T. W., Busby, S. J. W., & Browning, D. F. (2022). Inexpensive protein overexpression driven by the NarL transcription activator protein. Biotechnology and Bioengineering, 119, 1614– 1623. https://doi.org/10.1002/bit.28071

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block headercontent %}
![Logo](https://static.igem.wiki/teams/4436/wiki/results.png)
{% endblock %}
{% block page\_content %}

## CsgA Knockout

---

#### Transformation of PKD46 into MC4100 and MC4100-Z1

![LC99 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2038.jpg)

![LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2040.jpg)

![LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2042.jpg)

![LC99 + pKD46](https://static.igem.wiki/teams/4436/wiki/img-2039.jpg)

#### pkD3 PCR 8/1/22

![Gel Results](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-1.jpg)

lane 1: ladder, lane 2: pdk3 (control), lane 3/4: PCR amplicons

#### pKD3 PCR 8/3/22

![Black](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-black.jpg)

![White](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-white-1.jpg)

![Black 2](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-2-black.jpg)

![White 2](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-3-gel-1-white.jpg)

lane 1: ladder, lane 2: pdk3 (control), lane 3/4: PCR amplicons

#### pkD3 PCR 8/4/22

![pKD3-PCR-8_4](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-4-gel.png)

#### pkD3 8/4/22 trial 2

![PCR Cleanup Protocol](https://static.igem.wiki/teams/4436/wiki/pcr-cleanup.jpg)

## pkD3 Transformation

---

#### 8/4/22 transformation results

![Transformation Results](https://static.igem.wiki/teams/4436/wiki/screen-shot-2022-10-09-at-8-20-07-pm.png)

#### pkD3 PCR 8/5/22

![pKD3 PCR](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-5-1.png)

#### pkD3 transformation 8/5/22

![8/5 Transformation](https://static.igem.wiki/teams/4436/wiki/8-5-transformation.jpg)

#### pkD3 with DMSO 8/8/22

![pkD3 with DMSO](https://static.igem.wiki/teams/4436/wiki/pkd3-with-dmso-8-8.png)

#### pkD3 transformation 8/8/22

![Transformation](https://static.igem.wiki/teams/4436/wiki/8-8-transformation.jpg)

#### pkD3 PCR with DMSA 8/9/22

![PCR - pKD3 with DMSO 8/9](https://static.igem.wiki/teams/4436/wiki/pkd3-with-dmso-8-8.png)

#### pkD3 PCR w/ new primers 8/10/22

![pKD3 PCR with new primers](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-w-new-primers-8-10.png)

#### pkD3 PCR with old primers 8/10/22

![pKD3 PCR 8/10](https://static.igem.wiki/teams/4436/wiki/pkd3-pcr-8-10.png)

#### pKD3 transformation 8/11/2022

![LB + cm25 LC14](https://static.igem.wiki/teams/4436/wiki/lb-cm25-lc14.jpeg)

![LB + cm25 LC99](https://static.igem.wiki/teams/4436/wiki/lb-cm25-lc99.jpeg)

![LB LC14](https://static.igem.wiki/teams/4436/wiki/lb-lc14.jpeg)

![LB LC99](https://static.igem.wiki/teams/4436/wiki/lb-lc99.jpeg)

#### Successful transformation of LC14 and LC99 9/22/2022

![Transformation of LC14 and LC99](https://static.igem.wiki/teams/4436/wiki/transformaiton-of-lc14-and-lc99.jpg)

![LB LC99 + pkD46 and LC LC14 + pKD46](https://static.igem.wiki/teams/4436/wiki/lb-lc99-pkd46-and-lc-lc14-pkd46.jpg)

#### Colony PCR 10/5/22

![Colony PCR](https://static.igem.wiki/teams/4436/wiki/10-5-colony-pcr-2.png)

Lanes:

DNA ladder

MC4100 colony - negative control

MC4100 csgA KO colony

MC4100Z1 csgA KO colony

MG1655 csgA KO colony

MC4100 ackA KO colony

#### Successfully transformed pkD3 10/5/22

![Transformations](https://static.igem.wiki/teams/4436/wiki/img-1644.jpg)

{% endblock %}