iGEM 2021 — Team Pittsburgh

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# Team:Pittsburgh/Notebook

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* [**Safety**](https://2021.igem.org/Team%3APittsburgh/Safety)
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* **Outreach**
  + [Human Practices](https://2021.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education](https://2021.igem.org/Team%3APittsburgh/Education)* **Project**
    + [Description](https://2021.igem.org/Team%3APittsburgh/Description)
    + [Design](https://2021.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2021.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2021.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2021.igem.org/Team%3APittsburgh/Notebook)
    + [Results](https://2021.igem.org/Team%3APittsburgh/Results)
    + [Modeling](https://2021.igem.org/Team%3APittsburgh/Model)
    + [Implementation](https://2021.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2021.igem.org/Team%3APittsburgh/Contribution)
  * **Team**
    + [Team](https://2021.igem.org/Team%3APittsburgh/Team)
    + [Attributions](https://2021.igem.org/Team%3APittsburgh/Attributions)
    + [Collaborations](https://2021.igem.org/Team%3APittsburgh/Collaborations)
  * [**Home**](https://2021.igem.org/Team%3APittsburgh)

![](https://static.igem.org/mediawiki/2021/7/73/T--Pittsburgh--colon_and_detective.png)

## **Notebook**

Preliminary Research

May 3, 2020 - June 4, 2021; Everyone

Virtual meetings conducted to conceptually develop our project.

May 27, 2021; Everyone

Took inventory of lab supplies

Week 1

June 10, 2021; Everyone

Grew the streaked out plates for pMAL\_beta\_gal. Plasmid obtained from Dr. Xiaodong Zhu’s lab.

June 11, 2021; Angel & Isaac

Made liquid culture.

June 12, 2021; Everyone

Mini prep the pMAL-beta-gal

Week 2

June 15, 2021; Angel, Ethan, and Isaac

Ran a PCR for the pCZ10221 with 1% agarose gel at 120V for 45 minutes then extracted DNA from the gel.

![](https://static.igem.org/mediawiki/2021/1/1e/T--Pittsburgh--june15gel.png)

**Figure 1**. 6/15/21 PCR gel for pCZ10221

Week 3

June 22, 2021; Isaac, Kendall, and Madhu

Ran the PCR for pCZ10221

![](https://static.igem.org/mediawiki/2021/1/1e/T--Pittsburgh--june15gel.png)

**Figure 1**. 6/15/21 PCR gel for pCZ10221

June 23, 2021; Angel, Ethan, Isaac, and Kendall

Treated the PCR product with Dpn1, Made a 1x TAE buffer, Ran the 1% gel at 120V for 30 minutes, Extracted DNA from the gel, and the DNA concentration is 52.8 ng/uL

![](https://static.igem.org/mediawiki/2021/f/f7/T--Pittsburgh--june23gel.png)

**Figure 2**. 6/23/21 PCR gel for pCZ10221

Week 4

June 28, 2021; Angel, Isaac, and Madhu

Transformed pMAL\_beta\_gal into BL21 cells.

June 29, 2021; Isaac, and Kendall, and Madhu

The plate with just Amp+ grew but the IPTG/X-gal plate did not grow. Made liquid culture.

![](https://static.igem.org/mediawiki/2021/8/8d/T--Pittsburgh--june28_BL21colonies.png)

**Figure 3**. 6/28/21 BL21 colonies

June 30, 2021; Everyone

Made the glycerol stocks and liquid culture for plasmids from Dr. Robert Shank’s lab.

Week 5

July 6, 2021 - July 9, 2021; Angel

Designed pUC18\_grxA, pUC18\_katG, and pUC18\_dps which contain OxyR binding sites.

Week 6

July 12, 2021; Angel

Ordered the OxyR binding sites plasmids from genscript

Week 8

July 26, 2021; Everyone

Transformed pUC18\_grxA into Dh5 alpha and grew on Amp+ plates.

![](https://static.igem.org/mediawiki/2021/9/93/T--Pittsburgh--july26_Dh5%CE%B1_colonies.png)

**Figure 4**. 7/26/21 Dh5α colonies

July 30, 2021; Everyone

Ran initial H2O2 induction experiment using the lysis buffer. The blue pigment was not created from X-gal.

![](https://static.igem.org/mediawiki/2021/7/72/T--Pittsburgh--july30_lysedcells.png)

**Figure 5**. 7/30/21 Lysed cells following H2O2 exposure

Week 9

August 3, 2021; Angel, Emma, Ethan, Isaac, and Kendall

MiniPrep the pUC18\_grxA plasmid and ran a restriction digest.

![](https://static.igem.org/mediawiki/2021/9/90/T--Pittsburgh--aug3_digestgel.png)

**Figure 6**. 8/3/21 Restriction Digest Gel

August 4, 2021; Isaac, Kendall, and Madhu

Transformed pUC18\_grxA, pUC18\_katG, and pUC18\_dps into Dh5α cells, grew overnight culture

![](https://static.igem.org/mediawiki/2021/b/b8/T--Pittsburgh--aug4_Dh5%CE%B1_colonies.png)

**Figure 7**. 8/4/21 Dh5α colonies.

August 13, 2021; Angel, Ethan, and Isaac

MiniPrep the plasmids and ran restriction digestion.

![](https://static.igem.org/mediawiki/2021/4/44/T--Pittsburgh--aug6_digestgel.png)

**Figure 8**. 8/6/21 restriction digest gel

Week 10

August 8, 2021; Everyone

Ran H2O2 induction through incorporating the H2O2 into the agar plates. No blue colonies were detected.

Week 11

August 13, 2021; Kendall and Madhu

Ran H2O2 plate experiment again. No blue colonies.

![](https://static.igem.org/mediawiki/2021/9/99/T--Pittsburgh--aug13_Dh5%CE%B1_colonies.png)

**Figure 9**. 8/13/21 Dh5α cells on Amp+, X-gal, and

August 14, 2021; Madhu

Streaked out cells from Dr. Shank’s lab.

Week 12

August 25, 2021

Designed and ordered pRSF\_His\_FimH and pRSF\_SNAP\_His\_FimH from gen script.

Week 13

August 31, 2021; Madhu

Made BL21 competent cells.

September 3, 2021; Everyone

Transformed the pRSF\_His\_FimH and pRSF\_SNAP\_His\_FimH into BL21 pLysS cells.

![](https://static.igem.org/mediawiki/2021/3/32/T--Pittsburgh--sept3_BL21.png)

**Figure 10**. 9/3/21 BL21 cells on Kan+ plates

Week 14

September 6, 2021; Everyone

MiniPrep pRSF\_His\_FimH and pRSF\_SNAP\_His\_FimH plasmids and sent for sequencing.

September 7, 2021; Everyone

Ran H2O2 induction using a well plate and ONPG instead of X-gal. No yellow color was detected.

Week 16

September 26, 2021; Ethan and Isaac

Ran H2O2 induction using a well plate and ONPG instead of X-gal again. No yellow color was detected.

Week 17

October 4, 2021 - October 8, 2021; Ethan and Isaac

Continued to optimize H2O2 experiment.

Week 18

October 15, 2021; Angel and Madhu

IPTG on BL21 cells containing pRSF\_His\_FimH and pRSF\_SNAP\_His\_FimH. Then ran an SDS-PAGE gel.

Week 19

October 20, 2021; Kendall and Madhu

Performed nickel bead assay.

![](https://static.igem.org/mediawiki/2021/0/0e/T--Pittsburgh--logo.png)

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# Team:Pittsburgh/Engineering

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* [**Safety**](https://2021.igem.org/Team%3APittsburgh/Safety)
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    + [Design](https://2021.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2021.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2021.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2021.igem.org/Team%3APittsburgh/Notebook)
    + [Results](https://2021.igem.org/Team%3APittsburgh/Results)
    + [Modeling](https://2021.igem.org/Team%3APittsburgh/Model)
    + [Implementation](https://2021.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2021.igem.org/Team%3APittsburgh/Contribution)
  * **Team**
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## **Engineering**

Throughout the course of our project, many of our experimental plans underwent multiple design cycles. This process began with our plasmid designs. For the FimH plasmids, we needed to not only make sure to use a tag that would have a strong affinity for our marker of interest but also design a control plasmid to ensure accurate measurement of tag affinity. FimH is known to strong affinity to mannose[1](#citations), so we chose to use a histidine tag due to shown that chimera’s of FimH with a hexahistidine tag could be stimulated by binding to Ni-NTA beads. The SNAP tag can be attached to a protein of interest and functions to tag the targeted antibodies. FimH His and SNAP tags were designed in benchling for our experimental use.

We knew from the literature that FimH was connected the lacZ transcription factor OxyR[2](#citations), but figuring out the exact connection stumped us. Through additional research and a conversation with Dr. Robert Shanks, we discovered that FimH binding up-regulated OxyR transcription through the genes katG[3](#citations), dps[4,](#citations)[5](#citations), and grxA[6,](#citations)[7](#citations). Plasmids gene reporter assays were designed o regulate the expression lacZα using the Benchling website. We also discover that hypoxia can induce OxyR, so we began to design experiments to test gene transcription level in response to hypoxia akin to that of the tumor microenvironment.

Our H2O2 experiment required the most rounds of trouble shooting. We modeled our determination of critical H2O2 concentration after Rodríguez-Rojas et. al. and our lacZ assay after Schaefer et al.. In our initial run, we did not see any color change. We decided to add lysis buffer to optimally exposed the lacZ to the x-gal solution. However, our efforts still failed, and we had to go back to the drawing board. Through team discussions and guidance from our mentors, we decided to put the experiment on hold until we could determine the critical H2O2 concentration to ensure optimal cell impact. We determined this value to be 1mM. This concentration was subsequently used as a reference for 30-minute killing curves that show a clear dose-effect in survival rate.

We were still finding little success with this experiment, as we did not see a significant difference across varying concentrations of H2O2. We utilized a more involved strategy of sampling from culture tubes rather than our initial method of using a 96 well plate. The 96 well plates did not allow the bacteria to grow optimally due to poor volume of culture and not enough air. We made the cultures from frozen stock, grew them for 30 minutes to reach the exponential phase, then from this phase we started the timer and every 30 minutes took a sample from each of the concentrations of each of the plasmid types (8 samples at each interval total). The final procedure can be found in [**Experiments.**](https://2021.igem.org/Team%3APittsburgh/Experiments)

### References

1. Schwan, W. R.; Beck, M. T.; Hung, C. S.; Hultgren, S. J. Differential Regulation OfEscherichia Coli FimGenes Following Binding to Mannose Receptors. Journal of Pathogens 2018, 2018, 1–8. https://doi.org/10.1155/2018/2897581.
2. Shanks, R. M. Q.; Stella, N. A.; Kalivoda, E. J.; Doe, M. R.; O’Dee, D. M.; Lathrop, K. L.; Guo, F. L.; Nau, G. J. A Serratia Marcescens OxyR Homolog Mediates Surface Attachment and Biofilm Formation. Journal of Bacteriology 2007, 189 (20), 7262–7272. https://doi.org/10.1128/JB.00859-07.
3. Jung, I. L.; Kim, I. G. Transcription of AhpC, KatG, and KatE Genes in Escherichia Coli Is Regulated by Polyamines: Polyamine-Deficient Mutant Sensitive to H2O2-Induced Oxidative Damage. Biochemical and Biophysical Research Communications 2003, 301 (4), 915–922. https://doi.org/10.1016/s0006-291x(03)00064-0..
4. Antipov, S. S.; Tutukina, M. N.; Preobrazhenskaya, E. V.; Kondrashov, F. A.; Patrushev, M. V.; Toshchakov, S. V.; Dominova, I.; Shvyreva, U. S.; Vrublevskaya, V. V.; Morenkov, O. S.; Sukharicheva, N. A.; Panyukov, V. V.; Ozoline, O. N. The Nucleoid Protein Dps Binds Genomic DNA of Escherichia Coli in a Non-Random Manner. PLoS ONE 2017, 12 (8), e0182800. https://doi.org/10.1371/journal.pone.0182800.
5. Calhoun, L. N.; Kwon, Y. M. Structure, Function and Regulation of the DNA-Binding Protein Dps and Its Role in Acid and Oxidative Stress Resistance in Escherichia Coli: A Review. Journal of Applied Microbiology 2010, 110 (2), 375–386. https://doi.org/10.1111/j.1365-2672.2010.04890.x.
6. grxA - Glutaredoxin 1 - Escherichia coli (strain K12) - grxA gene & protein https://www.uniprot.org/uniprot/P68688 (accessed May 24, 2021).
7. Prieto-ÁlamoM.-J.; Jurado, J.; Gallardo-Madueño, R.; Monje-Casas, F.; Holmgren, A.; Pueyo, C. Transcriptional Regulation of Glutaredoxin and Thioredoxin Pathways and Related Enzymes in Response to Oxidative Stress. Journal of Biological Chemistry 2000, 275 (18), 13398–13405. https://doi.org/10.1074/jbc.275.18.13398.

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# Team:Pittsburgh/Experiments

![](https://static.igem.org/mediawiki/2021/2/26/T--Pittsburgh--logo_nobg.png)

* [**Safety**](https://2021.igem.org/Team%3APittsburgh/Safety)
* [**Parts**](https://2021.igem.org/Team%3APittsburgh/Parts)
* **Outreach**
  + [Human Practices](https://2021.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education](https://2021.igem.org/Team%3APittsburgh/Education)* **Project**
    + [Description](https://2021.igem.org/Team%3APittsburgh/Description)
    + [Design](https://2021.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2021.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2021.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2021.igem.org/Team%3APittsburgh/Notebook)
    + [Results](https://2021.igem.org/Team%3APittsburgh/Results)
    + [Modeling](https://2021.igem.org/Team%3APittsburgh/Model)
    + [Implementation](https://2021.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2021.igem.org/Team%3APittsburgh/Contribution)
  * **Team**
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  * [**Home**](https://2021.igem.org/Team%3APittsburgh)

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## Experiments

### Antibiotic Concentration

![](https://static.igem.org/mediawiki/2021/9/96/T--Pittsburgh--concentrations_antibiotic.jpg)

### SDS Page Gel

**1x SDS Running Buffer** (1L)

14.4 glycine

3.0g Tris Base

1g SDS

(fill to 1L with millipore dH2O)

**Coomassie Stain for Protein Gels (1L), store in dark**

60-80 mg Coomassie G-250

1 L MilliQ water; Stir for several hours to dissolve

**SDS Sample Buffer (3x); Store at -20°C**

1.5 mg Bromophenol Blue (BPB)

0.60 g SDS

3.0 mL GLycerol

3.9 mL 0.5 M Tris-HCl

1.5 mL β-mercaptoethanol

\_\_ mL dH2O to make final volume of 10 mL

**Expression Optimization Protocol**
1. Inoculate 50 mL of [antibiotic]+ LB-Miller overnight at 37°C in a 150 mL flask

1. 10 mL of overnight culture per L of large culture
2. Flask 3x larger than media volume
3. Approximately 5PM to 9AM

2. Inoculate large (1L) culture w/10 mL overnight culture

1. 11L with antibiotics (50 μg/mL AMP)
2. Begin at 9AM

3. Grow to OD600=0.5

1. Can use nanodrop or plate reader to measure
2. Blank with LB
3. Approximately Noon

4. Induce expression with 1mM [IPTG]final

1. [IPTG]final = 1 mL 1M IPTG/L culture
2. [IPTG]final = 1 mL 1M IPTG/L culture

5. Grow at 37°C for 3-4 hours

1. Grow at 37°C for 3-4 hours
2. Finish around 4PM

6. Take sample (1 mL) of cells, pellet at 21,000 xg for 1 minute
7. Resuspend pellet (1 mL sample) in 50 μL of denaturing sample buffer. Heat at 75-100°C for 5-10 minutes to lyse cells.
8. Shear DNA by vortexing and pellet again at 21,000 xg for 5 minutes before loading on a gel to test expression

1. Most likely 20% or 17% gel, 4-20% works as well

9. Run gel and spin 1 L cultures down in Nalgene 1 L bottles at 4000 xg, 10-15 minutes

1. Pour off liquid into sink (possibly with bleach)
2. Repeat for all of the same culture into the same bottle

10. For long term storage, resuspend in a resuspension buffer.

1. 50 mM HEPES-KOH pH8.0, 500 mM NaCl

11. Pellet resuspended cells into 50 mL tubes (conical) at 4000 xg, 10 minutes
12. Pour off liquid and freeze at -80°C until ready for purification

**PAGE + Coomassie Blue**
1. Rinse the gel in deionized water three times to remove any residual SDS- Soak the gel in enough Coomassie Blue to cover the surface and let sit for 10 minutes, shaking occasionally (or use a shaker table)
   - When you get tired of waiting for the gel to stain, place it in the microwave for 15 seconds at a time, being careful not to overheat the gel
   - Destain the gel by rinsing three times and then soaking in deionized water
   Replace solvent several times during the destaining process or add a kimwipe to absorb residual Coomassie Blue

### Preparation of Competent Cells

**Protocol**
1. Plate cells on LB plate. Pick an isolated colony and grow cells in 5 mL LB medium overnight
2. Transfer 1 mL culture to 100 mL LB media and grow at 37°C to OD600=0.4-0.6 in the morning; takes around 3 hrs
3. Place culture on ice for 5 min
4. Pellet cells at 4000 x g for 10 minutes
5. Resuspend cells in 1/10 volume of chilled TSB
6. Incubate on ice for 5 minutes
7. Subpackage into tubes (100 μL aliquots) and freeze in liquid nitrogen
8. Store at -80°C

### DNA Gel Electrophoresis

**Preparing Gel**1. Weigh 1g of agarose to 100 mL of 1x TAE buffer solution
2. Microwave and stir until fully dissolved (about 2 minutes)
3. When almost cooled, add 2 μL of ethidium bromide (careful)
4. Place combs, desired side down, and pour solution
5. For 50uL PCR product, use thicker side of comb

**Running Gel**1. Pour 1x TAE buffer till entire gel is covered
2. Load sample and ladder
3. Plug in the electrodes in a Negative to Positive direction
4. Run for 25 minutes at 125V
5. Turn off electrodes before removing cover
6. Look at gel under UV
7. Use a razor cut and transfer band of DNA to a clean eppendorf tube
8. Repeat for each column
9. Freeze at -20°C or continue onto gel purification protocol
10. Dispose of gel into specified ethidium bromide waste

### DNA Plasmid Experiments

**MiniPrep**1. Transfer 10 mL of cell culture, 1.5 mL aliquots at a time into 1 Eppendorf tube, using 1 tube for each culture.
2. Centrifuge for 1 min to pellet cells; discard the supernatant
3. Add 250uL of resuspension buffer (or water) and resuspend
4. Pipet up and down to fully resuspend cells
5. Add 350uL of Lysis solution
6. Invert 4-6 times
7. Add 350uL of neutralization solution
8. Invert 4-6 times
9. Centrifuge for 5 minutes to pellet cell walls and chromosomal DNA
10. Transfer supernatant to filter (blue)
11. Centrifuge for 1 minute
12. Discard flow through
13. Wash twice with wash buffer
14. Collect with sterile water

**Transformation**1. Combine 20 μL 5x KCM + x μL DNA (~ 10-100 ng) + ddH2O to 100 μL
2. Add 100 μL competent cells, mix gently and incubate on ice for 20 minutes
3. Heat shock at 42°C for 90 sec
4. Place on ice for 1 minute
5. Add 400-600 μL LB to cells
6. Shake for 45 min to 1 hour at 37°C
7. Plate 50-250 μL cell

### PCR

![](https://static.igem.org/mediawiki/2021/1/19/T--Pittsburgh--PCRtable1.jpg)

**Cycling Steps**

![](https://static.igem.org/mediawiki/2021/1/18/T--Pittsburgh--PCRtablecyclingsteps.jpg)

**H2O2 Gibson Assembly**1. Prepare the Gibson Assembly Master Mix as noted below protocol

1. Store at -20°C in 15 μL aliquots

2. Thaw a 15 μL aliquot of the Gibson assembly master mix and keep on ice until use
3. Measure the DNA concentration (ng/μL) of each assembly piece
4. Add 100 ng of linearized vector backbone and equimolar amounts of other assembly pieces to the thawed 15 μL master mix (see table below)
5. Incubate the assembly reaction at 50°C for 60 minutes, and then place on Ice
6. Transform 5 μL of the assembly reaction into 100 μL of competent DH5α E. coli

![](https://static.igem.org/mediawiki/2021/1/19/T--Pittsburgh--PCRtable1.jpg)

![](https://static.igem.org/mediawiki/2021/e/ee/T--Pittsburgh--gibsonassembly2.jpg)

### FimH Binding Experiments

**IPTG Induction**1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
2. Incubate the culture overnight at 37°C while shaking at 250 rpm.
3. Perform a 1:100 dilution of the overnight culture with 50 ug/mL of kanamycin and incubate the culture 3-4 hrs at 37°C while shaking at 250 rpm.
4. Read the absorbance of the culture via OD at 600 nm (it should be around 0.5-0.6).
5. Add IPTG to obtain a final concentration of 0.5 mM IPTG.
6. Incubate for 3-4 more hours at 37°C while shaking at 250 rpm.

**Nickel Bead Assay**1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
2. Prepare an additional falcon tube with 2 mL of LB broth and inoculate it with untransformed BL21 containing wild type fimH.
3. Incubate the cultures overnight at 37°C while shaking at 250 rpm.
4. Dilute all three cultures by a factor of 100 (1:100 dilution) in 2 mL of LB broth.
5. Add IPTG to all three cultures such that the final concentration of IPTG is 0.5 mM.
6. Add kanamycin stock (30 mg/mL) to the cultures with E.Coli cells containing the histidine tag and SNAP tag and histidine tag combination.
7. Grow the cultures for 2-4 hours until the O.D of the culture has reached 0.5-0.6. r.
8. Centrifuge the culture at 1400 rpm for 2 minutes and resuspend in 5 mL of PBS. Repeat 2-3 times to wash the bacteria.
9. Add 100 uL of the cultures to 3 separate microscope slides along with 40 uL of Ni-NTA beads.
10. View each slide under a microscope

**Yeast Agglutination Assay** 1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
2. Prepare an additional falcon tube with 2 mL of LB broth and innoculate it with untransformed BL21 containing wild type fimH.
3. Incubate the cultures overnight at 37°C while shaking at 250 rpm.
4. Pellet the cells by centrifuging at 14,000 rpm for 2 min. Completely remove the supernatant and resuspend the cells in PBS.
5. Prepare a glass microscope slide for each culture. Add 25 uL of the resuspended bacteria to each culture and 25 uL of Saccharomyces cerevisiae (yeast) suspended in PBS.
6. Start a timer and note the time at which agglutination begins.

**Imaging Protocol for Histidine and Histidine tag combination plasmids** 1. Inoculate 2 seperate falcon tubes containing 2 mL of LB broth and 50 ug/mL of kanamycin with BL21 bearing histidine and his/SNAP combination plasmids.
2. Inoculate a seperate falcon tube containing 2 mL of LB broth with BL21 cells containing native fim-H.
3. Incubate the culture overnight at 37°C while shaking at 250 rpm..

### OxyR Experimentation and Plasmid Design

**Designing Plasmids with OxyR Binding Sites**1. Delete the original promoter, operator, and ribosomal binding site (RBS).
2. Add in the different OxyR binding sites, which should be upstream of genes regulated by OxyR, including katG, dps, and grxA.

**H2O2 Growth Assay**1. Grow cells overnight. Inoculate the culture into 1.5 mL tubes and dilute it 1:50.
2. Add different concentrations of H2O2 to the tubes.
3. Measure OD600 with a plate reader.

**H2O2 Induction**1. Grow 2mL of cultures of the OxyR mutants and pUC-19 positive control overnight in 15 mL falcon tubes.
2. Dilute concentrate to 1:100
3. Create dilutions for H2O2

1. 1 mM
2. 500 μM
3. 250 μM
4. 100 μM
5. 500 μM

4. Incubate the diluted cultures at 37°C, shaking at 250 RPM, taking a sample of each every 30 minutes for 3 hours (0.2 mL H2O2:1.8 mL culture).
5. Pellet out the cells in the remaining liquid culture.
6. Freeze at -80°C if not running lacZ assay the same day.
7. Resuspend cells in lysis buffer and sonicate.
8. Lyse cells and run lacZ assay

**LacZ Assay**1. Prepare lysis buffer.

1. 60 mM Na2HPO4
2. 40 mM NaHPO4
3. 10 mM KCl
4. 1 mM MgSO4
5. 36 mM β-mercaptoethano
6. 166 μl/ml 7 lysozyme
7. 1.1 mg/ml ONPG
8. 6.7% PopCulture reagen

2. Add 120 μL of culture with 80 μL of lysis buffer.
3. Read absorbance at OD420.

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# Team:Pittsburgh/Results

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![](https://static.igem.org/mediawiki/2021/7/73/T--Pittsburgh--colon_and_detective.png)

## Results

## Fimbrial Mediated Attachment

The fimbrial cap protein fimH is the main anchoring element for our experimental design. Due to it's affinity to bind to surfaces, FimH has been frequently utilized in synthetic biology research as a tool to help localize bacterial species to surfaces such as mannose and nickel beads. Our project aims to modify the binding capabilities of FimH to allow it to anchor engineered E.coli cells to colorectal tumors. This is achieved through the use of protein tags, specifically the histidine and Synaptosomal- Associated Protein/histidine combination tags, which are intended to increase the binding affinity of the fimbriae to foreign surfaces such as a tumor microenvironment. The tags assist binding by allowing the FimH protein to effectively various target signals naturally expressed on the surface of tumors without conformational or binding strain.

## Transformation and Sequencing of Engineered Tags

In order to study fimbrial attachment mediated by protein tags, the team had to first successfully implement the tags in the E.coli strain BL21. Following plasmid design, transformations of the tags into E.coli cells were performed by the Genescript Biotech Corporation. To ensure that the correct plasmid DNA had been incorporated into the bacterial system, Sanger sequencing was performed on the bacteria.

## pRSF\_SNAP\_His\_FimH (SNAP + His Tag)   pRSF\_His\_FimH (His tag)

![](https://static.igem.org/mediawiki/2021/4/4d/T--Pittsburgh--SNAPSequence.png)
![](https://static.igem.org/mediawiki/2021/e/e2/T--Pittsburgh--HIStagsequence.png)

## Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis

Following transformations of the pRSF\_SNAP\_His\_FimH and pRSF\_His\_FimH plasmids into BL21 E.coli cells, cultures grown from the transformed bacteria were diluted to separate cultures with dilutions of 1:50 and 1:100. The cultures were then induced with Isopropyl β- d-1-thiogalactopyranoside (IPTG), an allolactose mimic, which is able to activate the expression of the FimH protein containing the Histidine and SNAP-Histidine tags. After induction, a sodium dodecyl sulphate–polyacrylamide gel electrophoresis, or SDS page gel was performed on the proteins. SDS page gels are separation techniques which use electrical currents to separate proteins based on their lengths. The basis of the separation is the higher mobility of smaller, shorter proteins compared to larger, bulkier proteins. In our case, we utilized separation based on protein length as a means of verification that the SNAP-Histidine and Histidine tags were properly expressed on the BL21's FimH. This was done by drawing comparisons between the measured lengths of proteins extracted from the transformed bacteria to the known lengths of the protein tags.

![](https://static.igem.org/mediawiki/2021/f/f1/T--Pittsburgh--SDSPageGel.png)

## Nickel Bead assay

Following transformations of the pRSF\_SNAP\_His\_FimH and pRSF\_His\_FimH plasmids into BL21 E.coli cells, a nickel bead assay was run on the cells. In the assay, bacterial cultures of the transformed cells were diluted 1:200 and induced with Isopropyl β- d-1-thiogalactopyranoside (IPTG) to activate expression of the modified FimH protein. Following induction, the cells were introduced to Ni (2+) NTA beads and then visualized under a microscope. A culture of untransformed BL21 was also run through the protocol alongside the modified BL21 as a negative control. The purpose of this assay was to compare fimbrial mediated attachment in E.coli cells containing native and modified fimbriae. Comparisions were drawn between the unmodified BL21 cells, BL21 cells containing histidine tagged fimbriae, and BL21 cells expressing fimbriae containing both a SNAP and a histidine tag. Binding was visualized using a standard laboratory microscope. Future imaging efforts may entail the use of electron microscopy and advanced visualization methods to view binding.

Localization of the modified fimbriae around the nickel beads should resemble the following.

![](https://static.igem.org/mediawiki/2021/9/91/T--Pittsburgh--Graphic.png)

Note: the above figure is not to scale and is depicted disproportionately to demonstrate the binding of modified fimbriae to the nickel bead surface.

## BL21 cells containing native fimbriae

![](https://static.igem.org/mediawiki/2021/thumb/4/4e/T--Pittsburgh--BL21.png/1184px-T--Pittsburgh--BL21.png)

## BL21 cells expressing fimbriae containing a Histidine tag

![](https://static.igem.org/mediawiki/2021/a/a5/T--Pittsburgh--His1.png)
![](https://static.igem.org/mediawiki/2021/5/53/T--Pittsburgh--His2.png)

## BL21 cells expressing fimbriae containing both SNAP and Histidine tags

![](https://static.igem.org/mediawiki/2021/0/09/T--Pittsburgh--SNAP1.png)
![](https://static.igem.org/mediawiki/2021/3/3f/T--Pittsburgh--SNAP2.png)

## Conclusions and Further Steps:

Sequencing results from BL21 cells indicated that the pRSF\_SNAP\_His\_FimH and pRSF\_His\_FimH were successfully taken up by BL21 cells during transformation. This indicated that the plasmid design was compatible with the cells. However, results obtained from the Nickel Bead assay and the SDS page gel indicate that the plasmid constructs need to be modified in the future to optimize binding and expression.

In the SDS page gel, the lengths of the protein are not clear from imaging taken of the gel. This error may be due to procedural errors such as inefficient IPTG induction conditions which may have reduced the expression of the fimH protein in the bacteria. However, another significant contributor may be inefficiences in the construct design which contribute towards inconsistent expression of the modified FimH protein. Going forward, we hope to optimize this protocol such that separation of proteins is efficient leading to clear visualization of beads such that the lengths of modified protein can be definitively determined.

In the Nickel Bead assay, the clear and black rods represent bacteria. Minimal binding of BL21 cells transformed with histidine tag and histidine-SNAP tag combination plasmids was observed during the assay. This is because on the microscope images, a low amount of rods can be seen clustering around the nickel beads. Rather, the majority of bacterial density appears to reside in the empty field outside of the bead area. While the number of rods may be affected by the design of the experimental protocol and the instrumentation used for visualization, the most likely explanation behind the low binding affinity is the design of the plasmids encoding for the protein tags. However, as the plasmid design is not finalized and can be altered, our team looks forward to optimizing protein tag function through continued iterations of the assay. For example by optimizing the buffer used in the experiment, any screening effects on the binding of bacteria to the Ni(2+)- NTA beads could be reduced. Other optimizations include varying the beads used in the experiments, such as the use of magnetic beads to allow for bound bacteria to be seperated from the solution and for binding to be quantified.

## Hypoxic Activation of OxyR and Reporter Release

## Hydrogen Peroxide Induction

The purpose of Hydrogen Peroxide Induction were two-fold. First, we were looking for the maximum concentration of hydrogen peroxide that would not inhibit the growth of our transformed bacteria. Secondly, we wanted to activate the OxyR protein in order to control the expression of the β-galactosidase enzyme.

It was found that different concentrations affected each OxyR controlled gene differently. To serve as a baseline, we were looking for the concentration at which the cells would achieve an OD of around 0.6. The most resistant gene was dps which was able to reach the OD in a max concentration of 1 micro molar. There was a tie between the KatG and grxA as the second most resistance transformed bacteria. KatG was the only gene to show a significant difference with increasing concentration.

![](https://static.igem.org/mediawiki/2021/c/ce/T--Pittsburgh--katGgraph.png)

### **Figure 1**. OD600 vs. time plot for katG

The exponential trendline gives us the doubling time for the culture at these concentrations.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Pittsburgh--katGtable.png)

### **Figure 2**. katG doubling times at H2O2 concentrations

The dps, grxA, and control plots are shown below.

![](https://static.igem.org/mediawiki/2021/7/72/T--Pittsburgh--dpsgraph.png)

### **Figure 3**. OD600 vs. time plot for dps

![](https://static.igem.org/mediawiki/2021/d/d9/T--Pittsburgh--grxAgraph.png)

### **Figure 4**. OD600 vs. time plot for grxA

![](https://static.igem.org/mediawiki/2021/4/49/T--Pittsburgh--pUC19graph.png)

### **Figure 5**. OD600 vs. time plot for pUC-19 control

# β- Galactoside Assay

The purpose of the β-galactosidase assay was to see if the β-galactosidase enzyme was produced and able to turn bacterial cells blue when in supplied with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal).

![](https://static.igem.org/mediawiki/2021/8/80/T--Pittsburgh--1mM_H202_grxA--Graphic.png)
![](https://static.igem.org/mediawiki/2021/b/bd/T--Pittsburgh--500uM_H202_grxA--Graphic.png)
![](https://static.igem.org/mediawiki/2021/c/c9/T--Pittsburgh--100_uM_H202_grxA--Graphic.png--Pittsburgh--500uM_H202_grxA--Graphic.png)
![](https://static.igem.org/mediawiki/2021/4/40/T--Pittsburgh--100_uM_H202_dps--Graphic.png)
![](https://static.igem.org/mediawiki/2021/2/21/T--Pittsburgh--100_uM_H202_katG--Graphic.png)

From left to right, the following plates were prepared to probe OxyR activity: 1 mM of H202 alongside DH5-α transformed with the grxA gene insert, 500 uM of H202 alongside DH5-α transformed with the grxA gene insert, 100 uM of H202 alongside DH5-α transformed with the grxA gene insert, 100 uM of H202 alongside DH5-α transformed with the dps gene insert, 100 uM of H202 alongside DH5-α transformed with the KatG gene insert. katG produced the most significant data.

The results of the β- Galactoside Assay were inconclusive as a color change was not able to be enacted in all transformed DH5-α cells (this includes those containing our designed OxyR gene inserts and those containing a puc19 control plasmid

# Conclusions and Further Steps:

To improve upon our H2O2 induction system, we plan to optimize the sequence for the OxyR binding site. We generated our original OxyR binding site plasmids by inserting the upstream sequences of genes controlled by OxyR (ex. grxA, dps, and katG). We aim to narrow down the sequence for the binding site to improve the affinity between OxyR and the specific sequence. Based on our modeling results, increasing the binding affinity would improve our system’s ability to detect changes in H2O2 concentrations. Furthermore, we want to use OxyR to control the expression of reporter genes other than lacZ-alpha. For instance, it may be possible to have OxyR induce the expression of proteins that are more easily detectable than beta-galactosidase.