iGEM 2020 — Team Pittsburgh

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# Team:Pittsburgh/Notebook

Bluetooth Bacteria

* [Safety](https://2020.igem.org/Team%3APittsburgh/Safety)
* [Parts](https://2020.igem.org/Team%3APittsburgh/Parts)
* Outreach
  + [Human Practices](https://2020.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education](https://2020.igem.org/Team%3APittsburgh/Education)* Project
    + [Description](https://2020.igem.org/Team%3APittsburgh/Description)
    + [Design](https://2020.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2020.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2020.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2020.igem.org/Team%3APittsburgh/Notebook)
    + [Proof of Concept](https://2020.igem.org/Team%3APittsburgh/Proof_Of_Concept)
    + [Results](https://2020.igem.org/Team%3APittsburgh/Results)
    + [Implementation](https://2020.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2020.igem.org/Team%3APittsburgh/Contribution)
  * Team
    + [Team](https://2020.igem.org/Team%3APittsburgh/Team)
    + [Attributions](https://2020.igem.org/Team%3APittsburgh/Attributions)
    + [Collaborations](https://2020.igem.org/Team%3APittsburgh/Collaborations)
  * [Home](https://2020.igem.org/Team%3APittsburgh)

## Notebook

Week -12 ~ 0

April 30, 2020 - July 24, 2020; Everyone

Virtual meetings conducted to conceptually develop our project, as the University of Pittsburgh research operations were remote until 07/24/2020.

Week 1

July 27, 2020; Sabrina & Angel

Took inventory of the lab.

July 29, 2020; Sabrina & Angel

Lpp-OmpA Streptavidin Transformation Attempt #1: plates did not have any colony growth, likely because it was only grown with ampicillin as antibiotic.

Week 2

August 4, 2020; Sabrina & Angel

Lpp-OmpA Streptavidin Transformation Attempt #2: the transformation of plasmid was successful due to growth on the non-antibiotic plate; however we require another attempt to figure out what antibiotics this plasmid is resistant to.

*Table 1. 8/4/20 Transformation results of plasmids Bba\_J36848 and PVS0043 after overnight growth at 37°C*

| Cell | Plasmid | Plate Media + Antibiotic | Growth |
| --- | --- | --- | --- |
| DH5alpha | Bba\_J36848 | LB + no antibiotic | Lawn/film formed |
| DH5alpha | Bba\_J36848 | LB + chlor | No |
| DH5alpha | Bba\_J36848 | LB + AMP | No |
| DH5alpha | Bba\_J36848 | LB + Kan | No |
| DH5alpha | PVS0043 | LB + AMP | Yes. Two small colonies formed |

![](https://static.igem.org/mediawiki/2020/a/ae/T--Pittsburgh--Plate1.jpg)

*Figure 1. Transformation results of plasmid Bba\_J36848 (streptavidin) after overnight growth at 37°C*

Week 3

August 10, 2020; Sabrina & Dara

Lpp-OmpA Streptavidin Transformation Attempt #3: unclear results because of a mistake made with the protocol (parafilm applied to plates when growing overnight).

*Table 2. 8/10/20 Transformation results of plasmid Bba\_J36848 after overnight growth at 37°C*

| Cell | Plasmid | Plate Media + Antibiotic | Growth |
| --- | --- | --- | --- |
| DH5alpha | Bba\_J36848 | LB + no antibiotic | Lawn/film formed |
| DH5alpha | Bba\_J36848 | LB + chlor | No |
| DH5alpha | Bba\_J36848 | LB + AMP | No |
| DH5alpha | Bba\_J36848 | LB + Kan | No |

August 11, 2020 ; Sabrina & Dara

Lpp-OmpA Streptavidin Transformation Attempt #3 (continued): Removed the parafilm from the plates to regrow again overnight; found that the backbone of the streptavidin plasmid is resistant to chlor.

*Table 3. 8/11/20 Transformation results of plasmid Bba\_J36848 after night at 37°C*

| Cell | Plasmid | Plate Media + Antibiotic | Growth |
| --- | --- | --- | --- |
| DH5alpha | Bba\_J36848 | LB + no antibiotic | Lawn/film formed |
| DH5alpha | Bba\_J36848 | LB + chlor | Yes, one large and one smaller colony formed |
| DH5alpha | Bba\_J36848 | LB + AMP | No |
| DH5alpha | Bba\_J36848 | LB + Kan | No |

![](https://static.igem.org/mediawiki/2020/4/4e/T--Pittsburgh--Plate2.jpg)

*Figure 2. Transformation plates from 9 AddGene plasmids grown in DH5alpha*

August 14, 2020; Dara

From the transformation confirmation plates of 9 AddGene plasmids in DH5alpha, and a streaked streptavidin plate, 11 total liquid cultures were made. The streptavidin cultures did not grow, and the 9 DH5alpha constructs were made into freezer stocks.

Week 4

August 17, 2020; Dara

Attempt #2 at growing liquid cultures from the streptavidin plasmid, but they did not grow.

August 18, 2020; Dara

Attempt #3 at growing liquid cultures for streptavidin and attempted to miniprep the culture for transformation, however it did not grow. Attempt #4 of the streptavidin liquid cultures were set to grow overnight.

August 19, 2020; Dara

Miniprepped streptavidin from overnight liquid cultures, and attempted transformations of AddGene plasmids into BL21 competent cells and streptavidin into BL21 and DH5alpha competent cells.

August 20, 2020; Dara

Transformation plates were checked, however all attempts were unsuccessful.

Week 5

August 25, 2020; Victor & Dara

Attempt #2 of AddGene plasmid transformation into BL21 competent cells.

![](https://static.igem.org/mediawiki/2020/5/5c/T--Pittsburgh--Plate3.jpg)

*Figure 3. 8/25/20 Transformation Plate Results of 9 AddGene plasmids grown in BL21*

August 26, 2020; Dara

Made liquid cultures from colonies on transformation plates of AddGene plasmids into BL21 cells. Made freezer stocks from those liquid cultures. Additionally, transformed streptavidin into DH5alpha and BL21 competent cells, and plated overnight.

![](https://static.igem.org/mediawiki/2020/b/b6/T--Pittsburgh--Plate4.jpg)

*Figure 4. 8/26/20 Transformation Plate Results of streptavidin plasmids grown in BL21 and DH5alpha*

August 28, 2020; Dara

After realizing nothing of value grew on the streptavidin transformation plates from 8/26/20, new liquid cultures were made of streptavidin. These cultures were miniprepped, transformed into DH5alpha and BL21 competent cells, and plated overnight.

Week 6

August 31, 2020; Angel

Liquid cultures of the 9 AddGene constructs were prepared and grown overnight.

September 1, 2020; Dara

Overnight liquid cultures of the 9 AddGene constructs (prepared on August 31) were induced for protein expression today

September 2, 2020; Sabrina

Liquid cultures of the GFP constructs (SW8765-Rosetta, and WSC1068-8W25113) were prepared and grown overnight.

September 3, 2020; Dara

From the overnight cultures of Rosetta and WSC1068, freezer stocks were made, and another round of overnight cultures (for the same constructs) were set up to express the proteins tomorrow.

Week 7

September 8, 2020; Dara

Inoculated and induced liquid cultures for expression of the 9 AddGene plasmids (using the overnight cultures prepared yesterday). Also ran a heating assay for the expressed constructs that were stored in –80 freezer.

September 10, 2020; Dara, Angel, & Sabrina

Prepared liquid cultures and the PCR machine for heat assay experiment.

Week 8

August 17, 2020; Dara

Attempt #2 at growing liquid cultures from the streptavidin plasmid, but they did not grow.

August 18, 2020; Dara

Attempt #3 at growing liquid cultures for streptavidin and attempted to miniprep the culture for transformation, however it did not grow. Attempt #4 of the streptavidin liquid cultures were set to grow overnight.

August 19, 2020; Dara

Miniprepped streptavidin from overnight liquid cultures, and attempted transformations of AddGene plasmids into BL21 competent cells and streptavidin into BL21 and DH5alpha competent cells.

August 20, 2020; Dara

Transformation plates were checked, however all attempts were unsuccessful.

Week 9

September 22, 2020; Dara

Liquid cultures grown to make more competent cells.

September 24, 2020; Dara

Liquid cultures were grown at 30 degrees Celsius from 1:40pm-6:45pm and left to grow further overnight in preparation for the Temperature Sensitive Dimer Induction experiment (measuring GFP expression). OD600 was measured in 1.5 hour intervals after induction start time. Last measurement was taken at 6:45pm (OD 0.018).

Week 10

October 1, 2020; Angel & Dara

We transformed constitutively mCherry expressing cells with the heat sensitive dimers.

Week 11

October 8, 2020; Angel & Dara

Re-plated the transformed bacteria on LB-Agar plates to confirm that the transformation was successful. Left overnight to incubate and grow at 37 degrees Celsius.

October 10, 2020; Sabrina

WSC1068 cells were transformed with heat-sensitive constructs after being incubated at 37°C overnight on LB-AMP/KAN plates. Liquid cultures were grown from colonies on plates and made into freezer stocks.

![](https://static.igem.org/mediawiki/2020/5/53/T--Pittsburgh--Plate6.jpg)

*Figure 5. Shows the transformation results of WSC1068 cells with nine heat-sensitive constructs after incubating at 37°C overnight. Colonies were growing on each LB-AMP/KAN plate. Single colony was selected for liquid culture and preparation of freezer stocks*

Week 12

October 13, 2020; Dara, Angel & Sabrina

Binding amine-coated magnetic nanoparticles (amine-MNP) to E. coli competent cells by suspending in solution.

*Table 4. OD change before and after addition of MNPs*

|  | Initial OD600 (no MNP added) | Final OD600 (MNP added and removed) |
| --- | --- | --- |
| DH5alpha | Bba\_J36848 | LB + no antibiotic |
| Sample 1 | 7.006 | 5.874 |
| Sample 2 | 1.886 | 0.561 |
| Sample 3 | 0.411 | 0.608 |
| Sample 4 (no cells, control) | 0.202 (blank) | 0.209 (blank) |

![](https://static.igem.org/mediawiki/2020/a/a6/T--Pittsburgh--Mag1.jpg)

*Figure 6. Separation of Amine-MNP Capture on 10/13; shows the amine-MNP suspended in E. coli solution. Based on visual observation, there was no clear magnetic separation between supernatant and MNPs. The supernatant extracted for absorbance measurement was cloudy and likely contained MNP*

October 14, 2020; Angel & Sabrina

Attempt #2 of binding amine-coated MNPs to E. coli competent cells.

![](https://static.igem.org/mediawiki/2020/e/ec/T--Pittsburgh--Mag2.jpg)

*Figure 7. Separation of Amine-MNP Capture (Sample 1-3) on 10/14/2020*

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Pittsburgh/Engineering

Bluetooth Bacteria

* [Safety](https://2020.igem.org/Team%3APittsburgh/Safety)
* [Parts](https://2020.igem.org/Team%3APittsburgh/Parts)
* Outreach
  + [Human Practices](https://2020.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education](https://2020.igem.org/Team%3APittsburgh/Education)* Project
    + [Description](https://2020.igem.org/Team%3APittsburgh/Description)
    + [Design](https://2020.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2020.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2020.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2020.igem.org/Team%3APittsburgh/Notebook)
    + [Proof of Concept](https://2020.igem.org/Team%3APittsburgh/Proof_Of_Concept)
    + [Results](https://2020.igem.org/Team%3APittsburgh/Results)
    + [Implementation](https://2020.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2020.igem.org/Team%3APittsburgh/Contribution)
  * Team
    + [Team](https://2020.igem.org/Team%3APittsburgh/Team)
    + [Attributions](https://2020.igem.org/Team%3APittsburgh/Attributions)
    + [Collaborations](https://2020.igem.org/Team%3APittsburgh/Collaborations)
  * [Home](https://2020.igem.org/Team%3APittsburgh)

## Engineering Success

In the short lab time made available to the team, a design cycle attempted to carry out was based on an imagined concept of a system that would induce gene expression in E. coli wirelessly. After a considerable amount of baseline information gathered from literature, and virtual conceptual development, the team was able to morph this idea into a full design: alternating magnetic fields, produced from a device, would be utilized to trigger a localized heating response from magnetic nanoparticles bound to the surface of E. coli. Within E. coli, the localized heating would activate temperature-sensitive dimers, initiating gene expression, made visible by fluorescent protein mWasabi.

Progress and results were recorded in small successes for the team, such as the successful transformation of temperature-sensitive plasmids pTlpA-Wasabi, pTlpA-Wasabi-NF, pDnaK-Wasabi, pTcI-Wasabi, pRpoH-Wasabi, pLaqI-Wasabi, pClp-Wasabi, PHtpG-Wasabi, and pGrpE-Wasabi, from the Mikhail Shapiro Lab, into BL21 and DH5alpha competent cells. These transformed plasmids became crucial for the team’s ability to conduct thorough heat assay experimentation. From the evidence presented on the [Results](https://2020.igem.org/Team%3APittsburgh/Results) page, observed fluorescence recorded through the conducted heat assays were induced from localized heating provided by controlled temperature environments, suggesting that heating theoretically produced from magnetic nanoparticles should be sufficient for the initiation of gene expression.

In addition to these findings, the team also managed to conjugate amine-functionalized magnetic nanoparticles (AF-MNPs) to the surface of the bacteria, suggested from experimentation found on the [Results](https://2020.igem.org/Team%3APittsburgh/Results) page. We observed that the OD reading of bacteria suspended in PBS decreased after the AF-MNPs were added and the nanoparticle-bacteria conjugate was removed; this suggested that the AF-MNPs were localized around the bacteria surface, which made the bacteria respond the magnet. If the AF-MNPs did not attach to the bacteria surface through electrostatic interaction, the OD reading should remain the same because the mass removed by the magnet would not contain bacteria.

Furthermore, we implemented plasmids with constitutive RFP expression to test the relative mWasabi expression levels. For these constructs, each cell will both constitutively express RFP and express mWasabi in response to thermal induction. This allows us to measure levels of fluorescence using a plate reader, microscopy, flow cytometry. Experiments were performed in triplicate to verify validity. This can be found on the [Results](https://2020.igem.org/Team%3APittsburgh/Results) Page.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Pittsburgh/Experiments

Bluetooth Bacteria

* [Safety](https://2020.igem.org/Team%3APittsburgh/Safety)
* [Parts](https://2020.igem.org/Team%3APittsburgh/Parts)
* Outreach
  + [Human Practices](https://2020.igem.org/Team%3APittsburgh/Human_Practices)
  + [Education](https://2020.igem.org/Team%3APittsburgh/Education)* Project
    + [Description](https://2020.igem.org/Team%3APittsburgh/Description)
    + [Design](https://2020.igem.org/Team%3APittsburgh/Design)
    + [Engineering](https://2020.igem.org/Team%3APittsburgh/Engineering)
    + [Experiments](https://2020.igem.org/Team%3APittsburgh/Experiments)
    + [Notebook](https://2020.igem.org/Team%3APittsburgh/Notebook)
    + [Proof of Concept](https://2020.igem.org/Team%3APittsburgh/Proof_Of_Concept)
    + [Results](https://2020.igem.org/Team%3APittsburgh/Results)
    + [Implementation](https://2020.igem.org/Team%3APittsburgh/Implementation)
    + [Contribution](https://2020.igem.org/Team%3APittsburgh/Contribution)
  * Team
    + [Team](https://2020.igem.org/Team%3APittsburgh/Team)
    + [Attributions](https://2020.igem.org/Team%3APittsburgh/Attributions)
    + [Collaborations](https://2020.igem.org/Team%3APittsburgh/Collaborations)
  * [Home](https://2020.igem.org/Team%3APittsburgh)

## Protocols

# Buffers & Solutions

# Antibiotic Concentrations

| Antibiotic | Stock Concentration (mg/mL) | Antibiotics vol for 20 mL LB plates (uL) | Antibiotics vol for 1 mL LB broth |
| --- | --- | --- | --- |
| Kan | 30 | 33.33 | 1 |
| Chlor | 30 | 20 | 0.67 |
| Strep | 50 | 12 | 0.60 |
| Amp | 100 | 20 | 0.50 |

# 1x SDS Running Buffer (1L)

1. 14.4 glycine
2. 3.0g Tris Base
3. 1g SDS
4. (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

# 5X KCM; Sterile filter, store at 4°C

| Final conc. | Stock conc. | Volume (ml) |
| --- | --- | --- |
| 0.5 M KCl | 2.5 M | 6 |
| 0.15 M CaCl2 | 1 M | 2.5 |
| 0.25 M MgCl2 | 1 M | 7.5 |
| ddH2O to | 30 mL total | 25 |

# 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

# Transformation Storage Buffer (TSB); Sterile filter and store at 4°C

| Final conc. | Stock conc. | Volume (ml) |
| --- | --- | --- |
| LB broth, pH 6.5 | 2x | 20.5 |
| 10% PEG 3350 | 50% | 10 |
| 5% DMSO | 100% | 2.5 |
| 10 mM MgCL2 | 1 M | 0.5 |
| 10 mM MgSO4 | 1 M | 0.5 |
| ddH2O to /td> 50 mL total | 15 mL | |

# Competent Cells

# Preparation of Competent Cells

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

# Preparation of Competent Cells

# PCR (Phusion, Q5 2x master mix)

| PCR Component | 1 | 5 | 9 | 11 |
| --- | --- | --- | --- | --- |
| 5X GC Buffer | 10 | 50 | 90 | 110 |
| dNTPs (10mM) | 1 | 5 | 9 | 11 |
| PCR Grade Water | 20 | 100 | 180 | 220 |
| DMSO | 1.5 | 7.5 | 13.5 | 16.5 |
| Betaine (5 M) | 13 | 65 | 117 | 143 |
| Sense Primer (10 μM) | 1.5 | 7.5 | 13.5 | 16.5 |
| Antisense Primer (10 μM) | 1.5 | 7.5 | 13.5 | 16.5 |
| Template DNA | 1 | 5 | 9 | 11 |
| Phusion DNA Polymerase (1 U/μL) | 0.5 | 2.5 | 4.5 | 5.5 |
| Total Volume | 50 | 250 | 450 | 550 |

| PCR Component | 1 | 5 | 9 | 16 |
| --- | --- | --- | --- | --- |
| 2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 |
| Forward Primer (10 μM) | 0.5 | 2.5 | 4.5 | 8 |
| Reverse Primer (10 μM) | 0.5 | 2.5 | 4.5 | 8 |
| Betaine (5 M) | 2.6.5 | 13 | 23.4 | 41.6 |
| DMSO | 0.5 | 2.5 | 4.5 | 8 |
| Water | 0.9 | 4.5 | 8.1 | 14.4 |
| Total Volume | 10 | 50 | 90 | 160 |

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25 cycles | 98°C   45-72°C   72°C | 5-10 seconds   10- 30 seconds   60- 120 seconds per kb |
| Final Extension | 72°C | 5-10 minutes |
| Hold | 4°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

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 supernatent
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

# Plasmid Screening (Colony PCR)

| PCR Component | 1 | 5 | 9 | 16 |
| --- | --- | --- | --- | --- |
| 2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 |
| Forward Primer (10 μM) | 0.5 | 2.5 | 4.5 | 8 |
| Reverse Primer (10 μM) | 0.5 | 2.5 | 4.5 | 8 |
| Betaine (5 M) | 2.6.5 | 13 | 23.4 | 41.6 |
| DMSO | 0.5 | 2.5 | 4.5 | 8 |
| Water | 0.9 | 4.5 | 8.1 | 14.4 |
| Total Volume | 10 | 50 | 90 | 160 |

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 seconds |
| 25 cycles | 98°C   45-72°C   72°C | 5-10 seconds   10- 30 seconds   60- 120 seconds per kb |
| Final Extension | 72°C | 5-10 minutes |
| Hold | 4°C |  |

# Preparation for Sequencing

* Set up sterile field
* Prepare 2 mL of LB per sample that you want to sequence; add 20 μL of culture to LB and add 2 μL of specified antibiotics
* Dilute to OD 0.4

# Microscope preparation (basic)

1. Add 10uL of sterile water to a pcr tube
2. Use a pipet-tip to transfer some cells from plate to the water by lightly scraping cells off the plate and swirling it in the water
3. Plate 2 μL of cells onto a microscope slide, place a slide cover over sample

# 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

# Protein Expression- 1L culture

# Expression Optimization Protocol

1. Inoculate 50 mL of [antibiotic]+ LB-Miller overnight at 37°C in a 150 mL flask
   * 10 mL of overnight culture per L of large culture
   * Flask 3x larger than media volume
   * ~5PM to 9AM
2. Inoculate large (1L) culture w/10 mL overnight culture
   * 1L with antibiotic (50 μg/mL AMP)
   * Begin at 9AM
3. Grow to OD600 =0.5
   * Can use nanodrop or plate reader to measure
   * Blank with LB
   * ~noon
4. Induce expression with 1mM [IPTG]final
   * [IPTG]final = 1 mL 1M IPTG/L culture
   * Aliquot into 1mL in 1.7 mL Eppendorf tubes
5. Grow at 37°C for 3-4 hours
   * 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
   * 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
   * Pour off liquid into sink (possibly with bleach)
   * Repeat for all of the same culture into the same bottle
10. For long term storage, resuspend in resuspension buffer.
    * 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

# Gel staining

# PAGE + Coomassie Blue

1. Rinse the gel in deionized water three times to remove any residual SDS
2. 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
3. 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.
4. Leave

# Temperature Sensitive Dimers

# Induction Protocol for Heat Sensitive Dimers (Piraner)

1. Pick a colony and grow overnight at 30C in 2xYT medium (what the heck is that? Gonna use LB)
2. In 20 hours (could also be adjusted to 14-16 hours), dilute to OD600 = 0.1 (approx. 30uL into 1 mL LB)
   * Use the dilution instead of measuring the OD
   * Place diluted culture back in 30 Celsius
3. After another 1 hour 15-30 min start checking culture OD600s
   * When ODs are 0.5-0.6, begin aliquoting
   * NOTE: different constructs grow at different rates, which can be tricky to coordinate; if there is an overshoot with the OD, you can re-dilute in more LB and try again
4. Aliquot 25uL E.coli into PCR tubes/strips (to give bacteria as much air as possible)
   * When moving culture to plate reader for OD measurement later, add 75uL LB, mix, and transfer 90uL into a 96 well plate
6. Incubate in the Thermocycler either at:
   * A set temperature
   * Gradient for 12 hours (this was the standard experimental time, but we can start seeing induction earlier)
7. Gradient for 12 hours (this was the standard experimental time, but we can start seeing induction earlier)

# Initial testing for GFP expression

1. Dilute to OD600 = 0.1 in LB (Sigma) with 100 μg/mL ampicillin for each culture and propagate to 30, 250 rpm for 1.5h. At OD600, dispense culture in 25 μL aliquots in PCR tubes.
Set up 3 PCR blocks (37°C, 42°C, 45°C respectively).

# Binding MNPs to E.coli surface

# Amine-functionalized MNPs

1. Thaw 1 tube of competent cells on ice. Add 200 uL of LB.
2. Spin down. Pipette out the supernatent. Add about 500 uL of PBS (10 mH, pH 7.0).
3. Check OD.
4. Add 50 uL AF-MNPs to the suspension for a final concentration of 0.5 mg mL-1.
5. Incubate for about 15 min at 250 rpm.
6. To measure how much of bacteria has been captured by the MNPs, use an external magnet to separate out the bacteria. Then, measure the OD600 of the supernatant.
7. After magnetic separation, resuspend the nanoparticle-bacteria conjugate in 100 uL of PBS.
8. Add 0.5 uL onto mesh carbon-coated copper grids.
9. After drying, the sample can be subjected to TEM analysis.

# Chitosan-functionalized MNPs

1. Dissolve 20 mg of chitosan to 1 M acetic acid with a final volume of 100 mL.
2. Add 70 mg of SPION to the solution.
3. Allow the solution to stir for 18 h.
4. This resulting solution should be a homogenous brown solution.
5. CS-MNPs can then be collected by magnetic separation and rinsed with water and ethanol to remove free chitosan.
6. Resulting CS-MNPs can then be dried at 60 °C under vacuum overnight.
7. Grow E.coli in LB media to log phase (OD 600 nm ~ 0.6).
8. Centrifuge the bacteria at 8000 rpm for 3 min and wash the cells several times with PBS.
9. Serially dilute the cells in PBS to obtain 105 CFU mL-1 suspensions.
10. Treat the cells with 200 μg mL-1 CS-MNPs for 6 h.
11. Centrifuge the mixture at 8000 rpm for 3 min and rinse it twice with PBS

# Biotinylated MNPs to Streptavidin on E.coli

1. Verify that streptavidin is expressed at the surface using biotinylated GFP.
2. Use E.coli that has already been transformed with streptavidin plasmid.
3. Dissolve biotinylated MNPs in solution.
4. Add the MNPs to suspension of E.coli.

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# Team:Pittsburgh/Results

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## Results

# Microscopy

We performed microscopy to measure the fluorescence per cell. This was done with samples that were incubated at 42 degrees for 12 hours. From these images, we noticed that TlpA and TcI worked only in some cells yet produced a very vibrant fluorescence. These were the heat-sensitive dimers. Alternatively, RpoH, Clp, HtpG, DnaK, GrpE, and LacIq are heat sensitive promoters. These produced more consistent fluorescence yet were not as bright. TlpA-NF contains a nonfluorescent mWasabi protein and thus, visualizing in the composite channel, produced the green background. No fluorescence was observed with this protein.

Further experiments may include computational analysis of fluorescence per cell and comparison to the different heat-sensitive strains to find the optimal mechanism for heating due to MNP excitation. Also, further microscopy using strains containing constitutive red fluorescence (mTangerine) to compare relative fluorescent protein expression.

# A. TlpA

![](https://static.igem.org/mediawiki/2020/0/00/T--Pittsburgh--oopipo.jpg)

# B. TlpA-NF

![](https://static.igem.org/mediawiki/2020/a/aa/T--Pittsburgh--oopiffpo.jpg)

# C. TcI

![](https://static.igem.org/mediawiki/2020/6/67/T--Pittsburgh--oopifdsffpo.jpg)

# D. RpoH

![](https://static.igem.org/mediawiki/2020/d/d8/T--Pittsburgh--oopifdsfdsffpo.jpg)

# E. HtpG

![](https://static.igem.org/mediawiki/2020/2/29/T--Pittsburgh--oopifdsfkhjkhdsffpo.jpg)

# F. Clp

![](https://static.igem.org/mediawiki/2020/3/3f/T--Pittsburgh--oopifdsddfsfdkhjkhdsffpo.jpg)

# G. DnaK

![](https://static.igem.org/mediawiki/2020/c/c7/T--Pittsburgh--oopifdsddfsfdsdfkhjkhdsffpo.jpg)

# H. GrpE

![](https://static.igem.org/mediawiki/2020/b/be/T--Pittsburgh--oopifdsddfkhjkhdsffpo.jpg)

# I. LacIq

![](https://static.igem.org/mediawiki/2020/8/8b/T--Pittsburgh--oopifdsdsgffgdfsfdsdfkhjkhdsffpo.jpg)

**Figure 1.**
Microscopy Images of 12-hour thermal induction of heat sensitive parts visualized using mWasabi expression: A-I: excitation and emission of mWasabi (green) and viewed using composite channel.

# mWasabi/mTangerine Thermal Assay

We performed 7-hour thermal inductions of our heat-sensitive constructs and analyzed the fluorescence using a plate reader. We measured the results in triplicate and calculated standard deviation and standard error for the measurements. We performed these experiments with cells that expressed mWasabi through heat-sensitive promoters and a separate constitutive mTangerine. This allows comparison of mWasabi output due to different temperatures. The graphs are calculated using fluorescence divided by OD to give an approximate fluorescence per cell.

Further directions include repeating the assay with thermal induction via AMF stimulation of MNPs. Also, including samples that were not exposed to heat to compare any potential leaky fluorescence output. Furthermore, I would like to take measurements at various different time points to measure rate of expression of the system and optimal thermal induction time.

# A

![](https://static.igem.org/mediawiki/2020/0/05/T--Pittsburgh--sdfsdfssdfrrsdfdfsdfsdfsdfsdfcxvxcvsfvvf.jpg)

# B

![](https://static.igem.org/mediawiki/2020/b/b7/T--Pittsburgh--sdfsdfssdfrrsdfdfsdfsffdfsdfsdfcxvxcvsfvvf.jpg)

# C

![](https://static.igem.org/mediawiki/2020/1/15/T--Pittsburgh--40Cnew.png)

**Figure 2.** 7-hour thermal incubation at 40 C of heat sensitive constructs (mWasabi) in cells that constitutively express mTangerine. Fluorescence measured by plate reader normalized using cell density (OD600) to give approximate fluorescence per cell. Error bars gives standard error. A. Heat sensitive promoter mWasabi expression fluorescence measurements. B. Constitutive mTangerine expression fluorescence measurements. C. Combined graph of mWasabi and mTangerine expression for comparison.

# A

![](https://static.igem.org/mediawiki/2020/2/27/T--Pittsburgh--sdffdfsdfsffdfsdfsdfcxvxcvsfjjkjbkvvf.jpg)

# B

![](https://static.igem.org/mediawiki/2020/a/ae/T--Pittsburgh--sdffdfsdfsffdfsdfsdfcxvxcddvsfjjkjbkvvf.jpg)

# C

![]( https://static.igem.org/mediawiki/2020/2/24/T--Pittsburgh--45C1new.png)

**Figure 3.** 7-hour thermal incubation at 40 C of heat sensitive constructs (mWasabi) in cells that constitutively express mTangerine. Fluorescence measured by plate reader normalized using cell density (OD600) to give approximate fluorescence per cell. Error bars gives standard error. A. Heat sensitive promoter mWasabi expression fluorescence measurements. B. Constitutive mTangerine expression fluorescence measurements. C. Combined graph of mWasabi and mTangerine expression for comparison.

# SDS-PAGE Gel

As another method of confirming that our protein was expressed, we ran our samples on a SDS-PAGE protein gel and stained it with Coomassie blue to confirm the presence of the mWasabi protein. Because the mWasabi expression isn’t able to be seen with the naked eye, this gel was used as a preliminary confirmation.

Future directions include running another SDS-PAGE gel after induction via AMF stimulation of MNPs. Also, running the gel with pre- and post- thermal induction of the samples to conclude that the band (and protein) appear in response to heat.

![](https://static.igem.org/mediawiki/2020/5/5c/T--Pittsburgh--sdfsdfssdfsdfdfsdfsdfsdfsdfcxvxcvsfvvf.jpg)

**Figure 4.**
Coomassie stained SDS-PAGE gel of post thermal induction heat sensitive constructs. Band observed at 26.9 kDa confirms that mWasabi was produced due to the thermal induction.

# Gradient Thermal Assay

We performed thermal induction assays with varying temperatures and culture agitations. We ran samples at 3 different temperatures, 37 C, 40 C, and 45 C. The cultures were either grown with shaking or in PCR tubes in a thermocycler. All the measurements are fluorescence normalized using OD/600 to determine relative fluorescence per cell. We observed increased expression of mWasabi with higher temperatures. Also, shaking tended to improve mWasabi expression, which would indicate that MNP attachment would be effective as it would be heating up the cells and not the media. There was no expression of mWasabi in TlpA-NF which is to be expected as this contains a non-fluorescent mWasabi.

![](https://static.igem.org/mediawiki/2020/9/9a/T--Pittsburgh--ffdfdsf.jpg)

**Figure 2.** Thermal Induction of heat sensitive constructs for 7-hours at varying time points. **Blue**: grown at 45 C in a larger culture while shaking. **Orange, Grey, Yellow**: grown at 37 C, 40 C, 45 C, respectively in separate 100 uL aliquots in a Thermocycler without shaking.

# Amine-functionalized MNPs (AF-MNPs) binding to E.coli Surface

We tested for the OD of bacteria suspended in PBS before and after AF-MNPs were added to the solution. We used PBS instead of LB to decrease the number of other surfaces, such as proteins, to which the AF-MNPs can non-specifically bind to. After incubating the bacteria with AF-MNPs, the resulting nanoparticle-bacteria conjugate was removed with a magnet. Since the OD of the solution decreased with AF-MNPs, we have some evidence that suggests that the AF-MNPs were binding to the bacteria.

Feature direction includes running a TEM imaging on the nanoparticle-bacteria conjugate to confirm that the AF-MNPs are localizing around the bacterial surface. Also, we plan to test the binding capabilities of other types of MNPs, such as ones attached to antibodies.

**Table 1.** OD change before and after addition of MNPs

|  | Initial OD600 (no MNP added) | Final OD600 (MNP added and removed) |
| --- | --- | --- |
| DH5alpha | Bba\_J36848 | LB + no antibiotic |
| Sample 1 | 7.006 | 5.874 |
| Sample 2 | 1.886 | 0.561 |
| Sample 3 | 0.411 | 0.608 |
| Sample 4 (no cells, control) | 0.202 (blank) | 0.209 (blank) |