iGEM 2018 — Team NDC-HighRiverAB

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# Team:NDC-HighRiverAB/Notebook

**HOME**

[NDC SYNTHETIC BIOLOGY](https://2018.igem.org/Team%3ANDC-HighRiverAB)

**PROJECT**

[DESCRIPTION](https://2018.igem.org/Team%3ANDC-HighRiverAB/Description)
[PARTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Parts)
[METHODOLOGY AND RESULTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/MethodologyandResults)
[MODELLING](https://2018.igem.org/Team%3ANDC-HighRiverAB/Model)
[SAFETY](https://2018.igem.org/Team%3ANDC-HighRiverAB/Safety)
[MEDAL CRITERIA](https://2018.igem.org/Team%3ANDC-HighRiverAB/MedalCriteria)
[FUTURE ASPIRATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Future)
[REFERENCES](https://2018.igem.org/Team%3ANDC-HighRiverAB/References)

**TEAM**

[TEAM MEMBERS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Team)
[SPONSORS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Sponsors)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Attributions)
[AWARDS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Awards)

**LAB**

[NOTEBOOK](https://2018.igem.org/Team%3ANDC-HighRiverAB/Notebook)
[PROTOCOLS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Protocols)

**OUTREACH**

[COLLABORATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Collaborations)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ANDC-HighRiverAB/Public_Engagement)
[HUMAN PRACTICES](https://2018.igem.org/Team%3ANDC-HighRiverAB/Human_Practices)

# Notebook

### Wednesday, July 4, 2018

Today we made sterile LB and some new agar plates (5 with amp, 1 without + some extra plates that should have had amp but didn't.) Ampicillin was added at a ration of 1 uL per mL of agar liquid.

We autoclaved some waste and some pipette tips.

We received competent cells from fredsense to use for a transformation with our NDC 001-1 in the PET21a plasmid.

We had an issue with rehydrating the DNA. We added 15 uL of distilled water to 4 ug of plasmid, but after centrifuging and stirring it seemed like there was very little DNA solution, so we added an additional 10 uL of water before proceeding with the transformation. We let this sit for 7 minutes before moving forward with the transformation.

Because of needing to rehydrate the DNA with additional water, the competent cells were thawed on ice, distributed into tubes, and then put back on ice before the DNA was added.

We followed the transformation procedure from the Synthetic Biology Workshop Protocol Manual (University of Lethbridge 2016).

The following plates were made:

| Plate | Competent Cells | Transformed with Plasmid | Ampicillin |
| --- | --- | --- | --- |
| Control | X |  |  |
| Control | X |  | X |
| Plate 1 | X | X | X |
| Plate 2 | X | X | X |
| Plate 3 | X | X | X |
| Plate 4 | X | X | X |

Present: Mrs. Côté, Ms. Larocque, Emily D, Emily H, Daisy H, Miguel L, Micha B, Kristina C, Mya G

### Thursday, July 5, 2018

2 colonies from each of Plates 1-4 (DH5a w/ NDCOO1-1 plasmid) were selected and placed in 5 mL LB with 5 uL ampicillin. These were left to incubate until tomorrow.

Present: Mrs. Côté, Mya G, Kristina C, Micha B

### Friday, July 6, 2018

Ms Larocque did subcultures of all 8 overnight cultures - 5uL AMP, 5mL LB, 50uL cells to grow from 10am - 1pm.

Ran mini-prep, restritction digest, and gel on overnight culture plate 1A and 2A.

- Gel may have cooled too much before pouring

- We poked through gel wells on first two rows

We are testing our bacteria with the nitrophenyl solutions.

Robert's Emails about steps:

1. Take a sample of the overnight culture (100-500 uL) and pellet it in the centrifuge (max speed, 5 mins).

2. Add detergent to the phosphate buffer to get a 1% detergent phosphate solution.

3. Add nitrophenol at a 1:50 dilution so that we end up with a 1 mM nitrohpenol, 1% detergent phosphate solution (this is the reaction mixture).

4. Resuspend the cell pellet in the reaction mixture and incubate at either room temp or 37 degrees at 200 rpm.

5. Take a sample (250 uL) at the desired time points and centrifuge it to remove the bacteria. (NOTE: incubate on ice for 10 mins before centrifugation if you can as this will help stop the reaction from progressing while you centrifuge). Once pelleted, take the solution and store it in a clear vial in the fridge.

6. When all samples are ready, take a picture with a white background to see the progression of the reaction at each timepoint. If the tubes get in the way of taking a good picture, use a piece of parafilm with the paper backing removed and put a 10 uL drop on it. Emily can show you how to do this (we used to do this in iGEM to mix loading dyes into samples).

Emily Hicks made the solutions needed and these were our steps to fit in between (steps 3-6 above)

1. Make reaction micture 2.5mL of nitrophenyl mixture (2 of them - 50uL of nitro, 2450uL of detergent)

2. Pick 4 cultures (2 overnight 3A and 2B, 2 subcultures 2A and 2B) and spin down:

a) 500uL

b) 1000uL

3. Resuspend pellets in 300uL of reaction mixture

4. Incubate at 37\*C

5. Check at 5 min = take 100uL out and put on ice for 10 min, centrifuge, check colour, take pictures)

6. Check at 10 min = take 100uL and repeat above

7. Check at 15 min

We didn’t end up removing the 100 uL samples as the reaction was happening very fast.

We didn't take an initial (0 min) picture for the nitrophenol oct - first picture was at 5 min.

Present: Emily Hicks (mentor), Emily H, Daisy H, Miguel, Micha, Mya, Mrs. Côté, and Ms. Larocque. (At various times throughout the lab).

### Thursday, September 9, 2018

Made 4 overnight cultures (from July 4 plate 3, colonies C, D, E, F) of DH5 alpha with NDC001-1 and Ampicillin.

Made 2 overnight cultures of plain DH5alpha.

Present: Mrs. Côté

### Monday, September 17, 2018

We took samples from the overnight cultures and made pellets from 500 uL of each by centrifuging for 5 min (4 samples contained NDC 001-1 and 2 were plain DH5alpha)

We prepared reaction mixture that contained 4410 uL of phosphate buffer, 490 uL of detergent (Triton), and 100 uL of either 4-nitrophenol octonoate or 100 uL of 4-nitrophenol palmitate (2 sets of reaction mixture).

We resuspended the pellets in 500 uL of reaction mixture (2 NDC 001-1 pellets with each reaction mixture, one plain DH5alpha with each), and also prepared control tubes with reaction mixture and no cells.

We incubated the tubes at 35-37 degrees C, photographing at the start and every 5 min.

The tubes with NDC001-1 and reaction mixture turned green. None of the other tubes turned green. The green seemed brighter for the octanoate than for the palmitate.

Present: Emily H, Daisy H, Sophia F, Mya G, Kristina C, Mrs Côté, Ms Laroque

### Wednesday, October 3, 2018

Made overnight cultures of E.coli and cells (5 of plain DH5alpha and 6 with our gene DH5alpha NDC 001-1 in the pET21a backbone)

 **MINIPREP**

3/6 miniprep tubes were unusable for one reason or another (one had an orange dot, we discarded this culture as it was thought to be contaminated)

From July 4 plate 3G (From tube smiley face from oct 2) Mini prep - when putting it into column, she did not take all liquid because it had dead cells floating that would not sink in centrifuge. Later, the column was dropped with our gene in it before ellution buffer. Kristina might not have put the entire ellution buffer (problem micropipetting?). We had to spin the wash buffer twice because we put the column into a mini centrifuge tube and the wash buffer could not all fit at the bottom.

From July 4 plate 3H (from tube 1 oct 2) Took some supernatant with dead cells, got all the liquid. We had to spin the wash buffer twice because we put the column into a mini centrifuge tube and the wash buffer could not all fit at the bottom.

This one took longer to do step 5 (spin down flakes)- had to redo it. But when we did the wash buffer we were able to use a bigger tube to collect the flow through and only had to spin it down once per wash.

 **RESTRICTION**

Finished restriction with Ecor1 and Psti with all 3 tubes of mini prep, and one tube of pSB1C3 linearized plasmid backbone. Restriction enzymes and buffers are expired.

 **LIGATION**

Completed Ligation as well. The temperature of the water bath was around 70 degrees C for the first 5-7 minutes.

Grew overnight cultures (2 with DH5alpha NDC 001-1 and 5 with plain DH5alpha in the pET21a backbone) for mini prep and transformation tomorrow

### Thursday, October 4, 2018

Added AMP to overnight cultures in the morning

Subcultured at 11am to prepare for transformation at 4pm

Group 1

**MINIPREP**

Did a mini prep on both overnight DH5alpha tubes with our NDC 001-1 gene in the pET21a backbone.

Sucked up a bit of the dead cells in the star tube

**RESTRICTION**

Finished restriction with Ecor1 and Psti with both tubes of mini prep, and one tube of pSB1C3 linearized plasmid backbone. Restriction enzymes and buffers are expired.

Might have put double buffer (4uL) into pSB1C3 tube because they did not see the first ammount come out and figured better safe than sorry. Also put in double Ecor1 for the same reason (2uL).

**LIGATION**

Completed Ligation as well.

Group 2

**TRANSFORMATION**

Followed procedure.

Made 4 tubes of competent cells; two tubes (ligation tube 1 and ligation tube 2), one with competent cell test kit (RFP), and one with nothing, just competent cells.

Plate positive control (competent cell test kit with RFP) plating on both with and without Chlor. Plating all ligation mixtures on chlor. Plating negative controls (competent cells) on both chlor and without chlor.

Ligation: Plate one plate with 300uL and one plate with 50uL for each tube

Competent cell kit (RFP): 50uL on each plate

Competent cell: 200uL on each plate

Expected results:

| Tube | Plate with Chloramphenicol | Plates without Antibiotic |
| --- | --- | --- |
| Ligation 1 | Growth | -- |
| Ligation 2 | Growth | -- |
| Competent Cell Kit (RFP) | Growth | Growth |
| Competent Cell | No Growth | Growth |

### Friday, October 5, 2018

Checked plates from last night. Incubator was plugged into a non-funcitonal plug. Switched plug but hours later still no cell growth except maybe on no antibiotic plate - not enough growth to be sure.

Made subculture from DH5a overnight cultures (100 uL into 10 mL) for transformation at 1pm

Cultured liquid samples from last night's transformations in ~7-8mL LB and incubated for 4-5 hours.

- 175uL of each ligation transformation used in LB with chlor

- 75 uL of positive control in LB with chlor

- 75 uL of negative control in LB with chlor

- 75 uL of positive control in LB with no antibiotic

- 75 uL of positive control in LB with no antibiotic

There was no growth in all of the chlor tubes, and only very little growth in the tubes without antibiotic which suggests our competent cells were good, but our plasmids did not get taken up.

Made plates (chlor and no antibiotic)

Made gel. Ran gel with restrictions from October 3. Gel was run at lower voltage than recommended because the machine was getting angry.

**Gel:**

| Lane | Contents |
| --- | --- |
| 1 | Ladder |
| 2 | 1 - restriction with E and P from NDC001-1 from July 4, 3H done October 3 |
| 3 | 2- restriction with E and P from NDC001-1 done from July 4, 3H done October 3 |
| 4 | 3 - restriction with E and P from NDC001-1 done from July 4, 3G done October 3 |

Made competent cells (had to run centrifuge longer for pellets - approximately 8 minutes total) and did transformation using ligations from yesterday (Star + Harry Potter).

One of the tubes of ligation mixture (not sure which) was thrown by accident - mechanical disturbance may affect results?

4 tubes of competent cells - one negative control, one positive control (orange tube from test kit), one star (L1), one harry potter (L2)

Plated transformed cells to grow overnight at 37C (same plate logistics as yesterday)

Gel results: no bands. Maybe because TAE buffer was too concentrated, maybe other reasons.

Present: Mrs. Côté and Ms. Larocque (AM) / Emily D, Kristina C, Mya G, Sophie F, Mrs. Côté and Ms. Laroque (PM)

### Saturday, October 6, 2018

Looked at plates from transformation - only positive and negative control from non antibiotic plates grew colonies. Our cells worked, but not our transformations, so the competent cells did not take anything up, or maybe our ligation did not work.

### Monday, October 8, 2018

Recieved our mini prep and performed restriction, ligation, and transformation with FREDsense.

Present: Emily Hicks (Mentor)

### Tuesday, October 9, 2018

Emily Hicks (Mentor)

Growth happened! All controls were as expected.

Picked colonies and grew in LB.

-----------

Went to University of Calgary to do some graphic design and lab work.

We learned about spectrophotometry and plated our cells to prepare for thursday's spectrophotometry lab. We learned a new way to streak our plates and new sterlization techniques.

Present: Emily Hicks (at FREDsense lab) / Jessica Larocque, Catherine Côté, Micha Bermas, Sophia Fraser, Mya George, and Emily Dudgeon, and well as Cassie Stillner from UofC iGEM and her TA Jason. (University of Calgary Lab)

### Wednesday, October 10, 2018

Took culture and tested with nitrophenol to make sure the gene and plasmid were in the culture, and then mini prepped.

Took miniprepped DNA and put it into wells 1A and 2A (2A in error) of the submissions kit.

Jessica Larocque sent the DNA to iGEM Headquaters.

### Tuesday, October 11, 2018

Jessica Larocque -
University of Calgary - Spectrophotometry lab.

Catherine Côté -
FREDsense - Modelling, judging form, registry page, wiki, poster, presentation


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# Team:NDC-HighRiverAB/Experiments

**HOME**

[NDC SYNTHETIC BIOLOGY](https://2018.igem.org/Team%3ANDC-HighRiverAB)

**PROJECT**

[DESCRIPTION](https://2018.igem.org/Team%3ANDC-HighRiverAB/Description)
[PARTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Parts)
[METHODOLOGY AND RESULTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/MethodologyandResults)
[MODELLING](https://2018.igem.org/Team%3ANDC-HighRiverAB/Model)
[SAFETY](https://2018.igem.org/Team%3ANDC-HighRiverAB/Safety)
[MEDAL CRITERIA](https://2018.igem.org/Team%3ANDC-HighRiverAB/MedalCriteria)
[FUTURE ASPIRATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Future)
[REFERENCES](https://2018.igem.org/Team%3ANDC-HighRiverAB/References)

**TEAM**

[TEAM MEMBERS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Team)
[SPONSORS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Sponsors)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Attributions)
[AWARDS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Awards)

**LAB**

[NOTEBOOK](https://2018.igem.org/Team%3ANDC-HighRiverAB/Notebook)
[PROTOCOLS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Protocols)

**OUTREACH**

[COLLABORATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Collaborations)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ANDC-HighRiverAB/Public_Engagement)
[HUMAN PRACTICES](https://2018.igem.org/Team%3ANDC-HighRiverAB/Human_Practices)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:NDC-HighRiverAB/Results

**HOME**

[NDC SYNTHETIC BIOLOGY](https://2018.igem.org/Team%3ANDC-HighRiverAB)

**PROJECT**

[DESCRIPTION](https://2018.igem.org/Team%3ANDC-HighRiverAB/Description)
[PARTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Parts)
[METHODOLOGY AND RESULTS](https://2018.igem.org/Team%3ANDC-HighRiverAB/MethodologyandResults)
[MODELLING](https://2018.igem.org/Team%3ANDC-HighRiverAB/Model)
[SAFETY](https://2018.igem.org/Team%3ANDC-HighRiverAB/Safety)
[MEDAL CRITERIA](https://2018.igem.org/Team%3ANDC-HighRiverAB/MedalCriteria)
[FUTURE ASPIRATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Future)
[REFERENCES](https://2018.igem.org/Team%3ANDC-HighRiverAB/References)

**TEAM**

[TEAM MEMBERS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Team)
[SPONSORS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Sponsors)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Attributions)
[AWARDS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Awards)

**LAB**

[NOTEBOOK](https://2018.igem.org/Team%3ANDC-HighRiverAB/Notebook)
[PROTOCOLS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Protocols)

**OUTREACH**

[COLLABORATIONS](https://2018.igem.org/Team%3ANDC-HighRiverAB/Collaborations)
[PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3ANDC-HighRiverAB/Public_Engagement)
[HUMAN PRACTICES](https://2018.igem.org/Team%3ANDC-HighRiverAB/Human_Practices)

# METHODOLOGY AND RESULTS

In order to reach our goal of breaking down fats, which are a major component of buildups in wastewater systems, we created a novel biobrick which would allow our bacteria to produce an esterase under control of an inducible-promoter.

### Design

* First, we researched an esterase-producing gene that could be expressed in E.coli. We decided to use the EstA gene, which is found in pseudomonas aeruginosa.
* For our promoter, we decided to use pLAC, which is lactose-inducible and is naturally found in E.coli.and was available in the registry (Part:BBa\_R0011).
* We chose the B0034 RBS (Part:BBa\_B0034).
* Finally, we chose the double terminator B0015, which is the most common terminator and is known to be reliable. (Part:BBa\_B0015).
* After we had chosen all of our biobrick parts, we had them synthesized together, creating our new biobrick: (Part:BBa\_K2694000).

![](https://static.igem.org/mediawiki/2018/3/3e/T--NDC-HighRiverAB--BioBrick.jpeg)

### Qualitative Testing

Once we received our DNA, we transformed it into DH5α E.coli. We then did a plasmid switch to clone our part into PSB1C3 for submission to the Registry. To test our new part, we grew up cultures of our bacteria overnight. We then subcultured them for three hours before beginning the testing.

We first did a qualitative test. From the literature, we found that we could use 4-nitrophenyl palmitate and 4-nitrophenyl octanoate to test our esterase activity. Both substrates, when cleaved by an esterase, would go from clear to green, allowing us to monitor the cleavage activity visually. We set-up a time-course assay where we mixed 1mM of each nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer. We also tested DH5⍺ cells without any plasmid as well as the ester mixture with no cells as controls. Figure 2. Shows the results from this experiment.

![](”imagefrompartspage”)
Figure 2: The top row shows centrifuge tubes containing the 4-nitrohenol ester reaction mixture for the palmitate (a) and octanoate (b) esters with no cells added. The middle row shows the response for a negative control where DH5⍺ cells without any plasmid were added to reaction mixtures containing 4-nitrophenyl-plamitate (c) or 4-nitrophenyl-octanoate (d). The bottom row shows the response for the BBa\_K2694000 circuit transformed into DH5⍺ cells when added to reaction mixtures containing 4-nitrohenyl-plamitate (e) or 4-nitrophenyl-octanoate (f). All reaction mixtures were 1mM of the nitrophenyl ester with 1% v/v Triton X-100 in 0.1M pH 7 phosphate buffer.

From these results, we concluded that our bacteria was indeed cleaving the substrates, resulting in the green color. The clear control remained in the controls, showing that it was our part specifically that was inducing this change.

### Quantitative Testing

Once we had shown that our bacteria could cleave the substrates to some extent, we wanted to try to show this more quantitatively. In collaboration with the University of Calgary’s iGEM team, we designed an experiment to test our reaction in a time course, using their spectrophotometer, in order to more accurately determine the color change.

.