iGEM 2018 — Team Waterloo

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# Team:Waterloo/Notebook

[Waterloo](https://2018.igem.org/Team%3AWaterloo)

* [Home](https://2018.igem.org/Team%3AWaterloo)
* Team
  + [Team](https://2018.igem.org/Team%3AWaterloo/Team)
* [Meet The Microbes](https://2018.igem.org/Team%3AWaterloo/Meet_the_Microbes)
* [Collaborations](https://2018.igem.org/Team%3AWaterloo/Collaborations)

- Project
  * [Description](https://2018.igem.org/Team%3AWaterloo/Description)
- [Design](https://2018.igem.org/Team%3AWaterloo/Design)
- [Experiments](https://2018.igem.org/Team%3AWaterloo/Experiments)
- [Interlab](https://2018.igem.org/Team%3AWaterloo/Interlab)
- [Demonstrate](https://2018.igem.org/Team%3AWaterloo/Demonstrate)
- [Results](https://2018.igem.org/Team%3AWaterloo/Results)
- [Notebook](https://2018.igem.org/Team%3AWaterloo/Notebook)
- [Parts](https://2018.igem.org/Team%3AWaterloo/Parts)
- [Safety](https://2018.igem.org/Team%3AWaterloo/Safety)
- [Attributions](https://2018.igem.org/Team%3AWaterloo/Attributions)
- Human Practices
  * [Human Practices](https://2018.igem.org/Team%3AWaterloo/Human_Practices)
- [Engagement](https://2018.igem.org/Team%3AWaterloo/Engagement)
- [Iat](https://2018.igem.org/Team%3AWaterloo/IAT)
- [Societal Considerations](https://2018.igem.org/Team%3AWaterloo/Societal_Considerations)
- Dry Lab
  * [Model](https://2018.igem.org/Team%3AWaterloo/Model)
- [Software](https://2018.igem.org/Team%3AWaterloo/Software)
- [Automation](https://2018.igem.org/Team%3AWaterloo/Automation)
- [Turbidostat](https://2018.igem.org/Team%3AWaterloo/Turbidostat)

# Notebook

## Waterloo iGEM 2018

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##

##

##

##

##

© 2018 Waterloo iGEM


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

# Team:Waterloo/Experiments

[Waterloo](https://2018.igem.org/Team%3AWaterloo)

* [Home](https://2018.igem.org/Team%3AWaterloo)
* Team
  + [Team](https://2018.igem.org/Team%3AWaterloo/Team)
* [Meet The Microbes](https://2018.igem.org/Team%3AWaterloo/Meet_the_Microbes)
* [Collaborations](https://2018.igem.org/Team%3AWaterloo/Collaborations)

- Project
  * [Description](https://2018.igem.org/Team%3AWaterloo/Description)
- [Design](https://2018.igem.org/Team%3AWaterloo/Design)
- [Experiments](https://2018.igem.org/Team%3AWaterloo/Experiments)
- [Interlab](https://2018.igem.org/Team%3AWaterloo/Interlab)
- [Demonstrate](https://2018.igem.org/Team%3AWaterloo/Demonstrate)
- [Results](https://2018.igem.org/Team%3AWaterloo/Results)
- [Notebook](https://2018.igem.org/Team%3AWaterloo/Notebook)
- [Parts](https://2018.igem.org/Team%3AWaterloo/Parts)
- [Safety](https://2018.igem.org/Team%3AWaterloo/Safety)
- [Attributions](https://2018.igem.org/Team%3AWaterloo/Attributions)
- Human Practices
  * [Human Practices](https://2018.igem.org/Team%3AWaterloo/Human_Practices)
- [Engagement](https://2018.igem.org/Team%3AWaterloo/Engagement)
- [Iat](https://2018.igem.org/Team%3AWaterloo/IAT)
- [Societal Considerations](https://2018.igem.org/Team%3AWaterloo/Societal_Considerations)
- Dry Lab
  * [Model](https://2018.igem.org/Team%3AWaterloo/Model)
- [Software](https://2018.igem.org/Team%3AWaterloo/Software)
- [Automation](https://2018.igem.org/Team%3AWaterloo/Automation)
- [Turbidostat](https://2018.igem.org/Team%3AWaterloo/Turbidostat)

# Experiments

## Inoculation

1. Aseptically pipette 5 mL sterile liquid broth into a test tube.
2. Add appropriate antibiotic, if required, to desired concentration using a pipette. Volumes depend on the specific antibiotic but there is a list adhered to the top door of the -20℃ freezer in the iGEM lab.
3. When pipetting the antibiotic, tilt the test tube so that the broth is close enough to the top of the tube that you can add the antibiotic to the broth. Dropping it in won’t work because you will typically be adding approximately 2.5 μL and the drop will be too small to fall.
4. If inoculating from frozen stock, work quickly! This is very important because if the cells thaw completely they will have a shorter lifetime in the freezer. Always keep frozen stocks on ice.
5. Take an autoclaved, and thus sterile, wooden inoculation stick and scrape some frozen stock from the tube and dip the stick into the liquid media and stir around.
6. Discard inoculation stick into another container, not the waste! Inoculation sticks are reusable because they can be autoclaved multiple times.
7. If inoculating from a plate, use a sterile wooden inoculation stick and touch a colony on the plate, then dip it into the liquid media and stir it around. Again, do not the discard sticks.
8. Place the tubes in a 37℃ incubator on the shaker for 12-24 hours. If possible, angle the tubes because it improves oxygenation of the culture as it shakes.

## Nanodrop

The NanoDrop (ND-1000) is a small-scale spectrophotometer that can detect DNA concentration and give an estimate of sample purity.

1. Open the nanodrop application on the computer and login to the iGEM account using the password “bio”.
2. Click on “Nucleic acid” then begin to initialize the unit.
3. Put some MilliQ water on a KimWipe and wipe down the pedestal and sampling arm.
4. Add 1.0 μL of ddH2O, then 1.0μL of elution buffer to blank.
5. Load 1.0 μL of sample and click “Measure” to determine the concentration and absorbance ratios. Record these below. Between trials, wipe the machine with a KimWipe. Make sure to leave machine clean and how you found it.

## Polymerase Chain Reaction (PCR)

1. Per reaction: In a 200 μL tube, mix:
   * 1 μL Forward Primer (10 μM)
   * 1 μL: ReversePrimer (10 μM)
   * 10 μL 2X Taq/Q5/Phusion Master Mix
   * 8uL Sterile, nuclease-free water
2. Add template individually to each sample. Template may be a few cells (i.e. picked colony, small volume of liquid culture), or extracted/purified DNA (i.e. plasmid, genome, linear). Typically, add 1 μL of ~1ng/uL extracted/purified DNA (dilute in nuclease-free water if needed) or 1 μL of overnight culture. For colony PCR, simply touch the colony with a sterile tip and mix that into your reaction tube.
3. Place samples in thermocycler.

   | Q5 | | Taq | | Phusion | || Step | Temperature (in C) | Time (min:sec) | Temperature (in C) | Time (min:sec) | Temperature (in C) | Time (min:sec) |
   | Initial denaturation | 98 | 0:30 | 95 | 0:30 | 98 | 0:30 |
   | Denaturation | 98 | 0:05 - 0:10 | 95 | 0:15 - 0:30 | 98 | 0:05 - 0:10 |
   | Annealing | 58-72 | 0:10 - 0:30 | 45-68 | 0:15 - 1:00 | 45-72 | 0:10 - 0:30 |
   | Extension | 72 | 0:20 - 0:30 per kb | 68 | 1:00 per kb | 72 | 0:15 - 0:30 per kb |
   | Final extension | 72 | 2:00 | 68 | 5:00 | 72 | 5:00 - 10:00 |
   | Hold | 4 | Infinity | 4 | Infinity | 4 | Infinity |

   * Annealing temperature depends on the primers you’re using.
   * Note that this is for standard PCR (where the template is extracted DNA). If your template is DNA in whole cells (i.e. colony PCR), then initial denaturation should be 5-10 min to lyse the cells.

## Plasmid miniprep

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps in the solution; it should have uniform turbidity.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
10. Add ≥ 30 μl DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

## Gel electrophoresis

*Setting up your rig:*

1. Usually we use the small rig with the corresponding small tray (holds a 50 mL agarose gel).
2. Tape the edges of the tray. This needs to be water-tight, as you will set your gel here, so then the liquid solution can solidify.

*Choosing a comb:*
There are several reasons you might want to run a gel, which correspond to different choices of comb.

1. If you are running a gel to visualize a result, use the comb with the smallest teeth, and use the side with the thinnest teeth. Usually you run a small amount of sample in a gel like this, so the wells don’t need to be large.
2. If you are running a gel for the purposes of gel extraction, use the comb with the largest teeth, and use the side with the thickest teeth. Usually you run a large amount of sample in a gel like this, so the wells need to be large.

*Pouring and running your gel:*

1. Add agarose into a flask to make the concentration of agarose 0.8-1.2% (e.g. 0.5 g agarose to make 50 mL 1% agarose gel)
2. Add desired amount of 1X TAE running buffer (50 mL for small tray, but use more if you are running a larger gel).
3. Microwave for 30 seconds. Using rubber mitt, vigorously swirl the partially-dissolved agarose in the flask.
4. Microwave for 15 seconds. Using rubber mitt, vigorously swirl the dissolved agarose in the flask. Visually inspect the solution to make sure there is no undissolved agarose particles floating in it. Keep microwaving (in short 15 second intervals or so) until fully dissolved.
5. Add Gel Red at 1:10 000 concentration (50 mL gel needs 5 μL gel red).
6. Pour gel into tray.
7. Put comb into place.
8. Let it sit for 20 minutes. Wait until gel loses some transparency and looks more “white” and cloudy.
9. Once the gel has set, remove the tape from the rig and replace the tray in the rig so the ends of the tray are open.
10. Add additional 1X TAE running buffer to the rig. Buffer should cover the top of the gel.
11. Carefully remove comb from gel.
12. Load samples and appropriate DNA ladder (generally a 1kb ladder is used).
13. Attach the lid to the gel rig and plug the lid into the electrophoresis machine.

## Gel extraction

1. Weigh microfuge tube.
2. Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 ml microcentrifuge tube and weigh the gel slice.
3. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the slice (1mg of gel : 4uL of buffer).

   * Note:
     + Calculation: weight of gel piece extracted = (weigth of gel in microfuge tube) - (weight of microfuge tube)
     + If the volume of the dissolved sample exceeds 800 μl, the loading of the sample onto the column should be performed in multiple rounds to not exceed the volume constraints of the spin column.
4. Incubate the sample between 37–55°C (typically 50°C), vortexing periodically until the gel slice is completely dissolved (generally 5–10 minutes).
5. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
6. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
7. Repeat wash (Step 6).
8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
9. Add ≥ 6 μl of DNA Elution Buffer to the centre of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

## DNA Purification

1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended.

   Sample type | Ratio of binding buffer : sample || dsDNA > 2 kb (plasmids, gDNA) | 2:1 |
   | dsDNA < 2 kb (some amplicons, fragments) | 5:1 |
   | ssDNA (cDNA, M13) | 7:1 |
2. Insert column into collection tube and load sample onto column. Spin for 1 minute, then discard flow-through.
3. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discard flow-through.
4. Repeat wash (Step 3).
5. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step.
6. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

## Heat shock transformation

1. Acquire ice and enough competent cells for your experimental samples and controls.
2. Thaw competent cells on ice.
3. Get new 1.5 ml tubes, and label them with your different sample names. One tube will be a positive control (e.g. uncut vector alone) to test if your transformation technique worked.
4. Add 50 μL of competent cells to each labelled tube and then add your DNA. If DNA is from a ligation, add the entire volume into the tubes. If it is from a miniprep, usually add 1-10 μL depending on the DNA concentration.
5. Incubate on ice for 30 minutes.
6. Heat shock at 42℃ for 45 seconds and then immediately put the tubes back onto ice.
7. Incubate on ice for 5 minutes.
8. Add 750 μL of LB broth to each tube then incubate at 37℃ for 45 minutes - 1 hour with shaking.
9. After incubation you will plate the cells onto selective media so only transformants will grow.

## Frozen stock preparation

1. Prepare an overnight culture of the cells that are to be made into frozen stock.
2. Aliquot 1 mL of overnight culture into a 1.5 mL microfuge tube.
3. Pellet cells by spinning for 1 minute at 16,000 x g. Discard supernatant.
4. Resuspend in 1 mL fresh liquid broth.
5. In a 1.5 mL cryovial tube, add 150 μL of stock glycerol solution.
6. To that cryovial tube, add 850 μL of freshly resuspended cells (from step 4).
7. Pipette up and down to mix.
8. Label cryovial tube appropriately and update the strain list.
9. Store in -80°C freezer.

## Competent cell preparation

1. Inoculate 99 mL of LB with 1000 μL of overnight culture and grow at 37°C to an OD of 0.300-0.400 (such as 0.368).
2. Place cells on ice once they reached an OD of 0.300-0.400 (such as 0.368) and chill for 20-30 minutes (swirling periodically for even cooling).
3. After 20 minutes, split culture into 2 cold Falcon tubes. There should be 50 mL in each then be centrifuged at 4°C for 15 minutes 13000g.
4. Decant LB and resuspend each in 10 mL cold MgCl2.
5. Centrifuge for 15 minutes at 4°C at 13000 x g.
6. Decant and re-suspend cells in each Falcon tube in 10 mL of ice cold CaCl2 and allow to sit on ice for 20 minutes of incubation.
7. Centrifuge for 15 minutes at 4°C and 2000 x g (this was done twice because the pellet was not fully formed).
8. Decant and re-suspend in 5 mL cold CaCl2 + 15% glycerol.
9. Aliquot l00-200 μL into prechilled sterile 1.5mL microfuge tubes on ice.
10. Flash freeze tubes using liquid nitrogen and place in -80°C freezer.

## Methionine sharing

1. Prepare an overnight culture of methionine non-producers and optogenetic methionine producers in the turbidostat.
2. Spin down 650 µL of the optogenetic population at 13000 RPM for 1 minute.
3. Remove the supernatant.
4. Resuspend cells in 25 mL of M9 media that lacks methionine (M9-met).
5. Spin down at 13000 RPM for 1 minute.
6. Aspirate the media.
7. Resuspend in 25 mL of M9-met with 25 µL of appropriate antibiotic for your population’s resistance.
8. Grow until OD nears 0.4.
9. Set aside 50 µL of the optogenetic population.
10. Spin down the rest at 13000 RPM for 2 minutes.
11. Aseptically filter sterilize the supernatant.
12. Take 100 µL of methionine non-producer culture, spin down 13000 RPM for 1 minute.
13. Draw off supernatant.
14. Resuspend in 1 mL of M9-met.
15. Set up experimental tubes and controls according to the following table.

| Test Tube #</ Control</ Components</  | | | | | |
| --- | --- | --- | --- | --- | --- |
| 1 | Experimental | → M9 - met with CcaS/R v2.0 JT2 removed (3 mL)   → Empty JT2 (50 µL) |
| 2 | Positive | → M9 - met with CcaS/R v2.0 JT2 removed (3 mL)  → CcaS/R v2.0 JT2 (50 µL) |
| 3 | Positive | → M9 - met with CcaS/R v2.0 JT2 removed (3 mL)  → Empty JT2 (50 µL)  → Met (150 µL) |
| 4 | Negative | → M9 - met with CcaS/R v2.0 JT2 removed (3 mL) |
| 5 | Negative | → Stock M9 - met (3 mL)  → Met (150 µL) |
| 6 | Negative | → Stock M9 - met (3 mL)  → Empty JT2 (50 µL) |
| 7 | Positive | → Stock M9 - met (3 mL)  → Empty JT2 (50 µL)  → Met (150 µL) |

16. Grow overnight at 37C
17. Record OD readings

## Growth Curves

### 1. Inoculating cells

* Inoculate cells into LB medium with appropriate antibiotics for selection
* The next morning re-inoculate from the overnight culture into M9 media with the appropriate antibiotics

### 2. Adjusting Cells to Light

* Once the cells have reached an OD of > 0.6 re-inoculate 100ul of culture into 35mL of M9 drop out media (-methionine)
* Place cells into the turbidostat with a stir bar
* Incubate at 35 degrees with stirring under green light for 3h
* NOTE: The intensity of green light should be the same intensity you want to use for the growth curve

### 3. Growth Rate Measurement

* After 3h of adjusting the cells to the green light begin taking OD 600 measurements every 20 min until the cells reach an OD of ~0.8
* Plot the ln(OD 600 values) vs Time
* Calculate the growth rate by taking the slope of the linear portion of the curve

## Waterloo iGEM 2018

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© 2018 Waterloo iGEM

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

# Team:Waterloo/Results

[Waterloo](https://2018.igem.org/Team%3AWaterloo)

* [Home](https://2018.igem.org/Team%3AWaterloo)
* Team
  + [Team](https://2018.igem.org/Team%3AWaterloo/Team)
* [Meet The Microbes](https://2018.igem.org/Team%3AWaterloo/Meet_the_Microbes)
* [Collaborations](https://2018.igem.org/Team%3AWaterloo/Collaborations)

- Project
  * [Description](https://2018.igem.org/Team%3AWaterloo/Description)
- [Design](https://2018.igem.org/Team%3AWaterloo/Design)
- [Experiments](https://2018.igem.org/Team%3AWaterloo/Experiments)
- [Interlab](https://2018.igem.org/Team%3AWaterloo/Interlab)
- [Demonstrate](https://2018.igem.org/Team%3AWaterloo/Demonstrate)
- [Results](https://2018.igem.org/Team%3AWaterloo/Results)
- [Notebook](https://2018.igem.org/Team%3AWaterloo/Notebook)
- [Parts](https://2018.igem.org/Team%3AWaterloo/Parts)
- [Safety](https://2018.igem.org/Team%3AWaterloo/Safety)
- [Attributions](https://2018.igem.org/Team%3AWaterloo/Attributions)
- Human Practices
  * [Human Practices](https://2018.igem.org/Team%3AWaterloo/Human_Practices)
- [Engagement](https://2018.igem.org/Team%3AWaterloo/Engagement)
- [Iat](https://2018.igem.org/Team%3AWaterloo/IAT)
- [Societal Considerations](https://2018.igem.org/Team%3AWaterloo/Societal_Considerations)
- Dry Lab
  * [Model](https://2018.igem.org/Team%3AWaterloo/Model)
- [Software](https://2018.igem.org/Team%3AWaterloo/Software)
- [Automation](https://2018.igem.org/Team%3AWaterloo/Automation)
- [Turbidostat](https://2018.igem.org/Team%3AWaterloo/Turbidostat)

# Results

# Methionine Sharing Experiment

 ![](https://static.igem.org/mediawiki/2018/2/23/T--Waterloo--resultssummaryfigure1versionfinal.png)

Multiple trials went underway for the growth of empty JT2 in knockout MetE media to definitively confirm that the predicted results occured in regards to CcaS/R JT2 and empty JT2 growth. As the goal of the experiment was to assure that there was no “leaking” of methionine from the CcaS/R JT2 cells when stimulated with methionine production, leaking that could support the growth of another population. The following test matrix was performed to assess the possibilty.

| Test Tube # | Control | Components | Results |
| --- | --- | --- | --- |
| 1 | Experimental | → M9 - met with CcaS/R JT2 removed (3 mL)  → Empty JT2 (50 µL) | OD = 0.010 |
| 2 | Positive | → M9 - met with CcaS/R JT2 removed (3 mL)  → CcaS/R v2.0 JT2 (50 µL) | OD = 0.820 |
| 3 | Positive | → M9 - met with CcaS/R JT2 removed (3 mL)  → Empty JT2 (50 µL)  → Met (150 µL) | OD = 0.799 |
| 4 | Negative | → M9 - met with CcaS/R JT2 removed (3 mL) | OD = -0.011 |
| 5 | Negative | → Stock M9 - met (3 mL)  → Met (150 µL) | OD = 0.004 |
| 6 | Negative | → Stock M9 - met (3 mL)  → Empty JT2 (50 µL) | OD = 0.005 |
| 7 | Positive | → Stock M9 - met (3 mL)  → Empty JT2 (50 µL)  → Met (150 µL) | OD - 0.812 |

In this chart, a negative control refers to an experiment where there was an expectation that there should be no bacterial growth as per our hypothesis, and a positive control denotes an experiment where there should have been growth expected.

As can be seen from the chart, the experimental trial, in which the CcaS/R JT2 strain was grown and removed from the methionine knockout media, had no growth once empty JT2 was introduced into it. This was an encouraging result as it suggested that there was little or no “leaking” of methionine produced by the CcaS/R JT2 during growth into the media, and thus the empty JT2 did not have any means to grow.

The minor growth of CcaS/R JT2 as a positive in control in trial 2 was expected, and was done to confirm that there would be no adverse factors from the removal of CcaS/R JT2 that were causing the introduced empty JT2 to not be able to grow, and that the composition of the media was static.

Trial 3 showed with the repliaction of experimental conditions and the addition of MetE that the reason that the growth was not happening in experimental conditions was in fact the lack of methionine in the M9 media. This showed growth as expected.

Trial 4 indicated no growth as expected. This was carried out, with the removal of the CcaS/R JT2 and addition of the antibiotic, to confirm that all the bacteria was being taken out and that no other bacterial contamination was what was being seen in the positive control tests that had reported growth.

Trial 5 similarly was carried out with stock M9 to assure no environmental contamination was in the media from the beginning, especially bacteria that required external methionine to grow.

Trial 6 was carried out as a negative control to assure that the stock media itself was not contaminated with any nutrients that could be used by the empty JT2 to grow. This was important as it is possible that trace amounts could have been consumed in the other trials by the CcaS/R JT2 bacteria and thus were not reflected in the experimental trial.

Finally, the 7th trial indicated that the empty JT2 strain that was in this bacteria was healthy and could grow in the desired conditions within the M9 media, with the methionine and the KM from stock solution.

# GFP vs Non-Fluorescent Flow Test Experiment

Our comparison of the flow cytometer against pour plating started with a fluorescent and a non-fluorescent culture of roughly the same OD values. Below our initial ODs and results from both the flow cytometer and pour plating.

|  | Fluorescent | Non-Fluorescent |
| --- | --- | --- |
| Initial OD | 0.405 | 0.430 |
| Flow Cytometer   480nm Laser at 10mW | 52% | 48% |
| Pour Plating   Percent total CFU/mL | 52% | 49% |

These results lead us to believe that the flow cytometer is representative of pour plating, indicating that the flow cytometer is most likely capable of giving accurate readings of a 50/50 population of fluorescent/non-fluorescent cells.

# MetE Metabolic Load Experiment

Four sample tubes of JT2 CcaS/R-containing cells in M9 were made, and all were placed in the turbidostat. The samples were made by inoculating the cells off a plate into complete M9 made with casamino acids as the amino acid source.

Two samples were grown under only red light, and two under only green light. The samples were optically insulated from each other. The cells were allowed to grow for 3 hours to acclimatize them to the light conditions they were being kept under.

Unfortunately, though the doubling times for the two green-light samples were 53.8 and 55.8 minutes, the doubling times for the samples grown under red light were 45.3 and 61.2 minutes. The red-light sample doubling times were too far apart to be averaged reasonably, and therefore no conclusion could be drawn from the experiment.

Additionally, the team realized that the metabolic load of MetE may be different in media made with casamino acids versus media made with an amino acid mixture without methionine. Therefore, the experiment was not repeated and estimates of metabolic load would henceforth only be obtained by observing growth rates under different light conditions in M9 made without methionine.

See pages 77 to 79 in the online lab book for additional details.

# “Robot” Experiments

In order to automate experiments and make the designed system more useable, the team set out to create a "robot" capable of measuring cell fluorescence (and hence co-culture composition). See the "Automation" page for full details.

# Single Population Behaviour Experiments

For our proof of concept, we wanted to use two populations of E. coli: slow growing DH5alpha with a GFP marker and light controllable JT2 with CcaS/R. Before creating a co-culture with these two populations, we needed to determine how they behave on their own.

## Light Control Experiments:

Before trying to create a stable co-culture, we had to confirm that we could control the growth of a single population of E. coli. This was done by replicating the experiments from the paper.

In order to replicate the experiments from this paper we had to develop a new protocol that would work for the equipment in our lab. This took many trials and troubleshooting until we developed our final protocol (please refer to our experiments page for more information on this)

### Results:

#### JT2 Ccas/R Growth rate in Response to Changes in Green Light

Below is a graph showing the growth rate of JT2 Ccas/R under three different intensities of green light, 24%, 48.8% and 100%. Increased green light exposure resulted in increased growth rate. This is what we expected to see based on the results from the paper.

##### Note on light set up:

**100% green light:** green light is on 100% of the time during the span of the experiment

**48.8% green light:** green light is on 48.8% of the time during the span of the experiment

**24% green light:** green light is on 24% of the time during the span of the experiment

 ![](https://static.igem.org/mediawiki/parts/5/58/Growth-rate-JT2-dec7.png)

Each Point represents the growth rate of a single tube of JT2 Ccas/R under 24%, 48.8% OR 100% green light. Growth rate was measured in duplicates for each light intensity.

#### GFP Expression in response to green light

We wanted to dig a little further into the cell’s response to changes in light intensity. To do this we measured the level of GFP expression for each population under different intensities of green light (using a flow cytometer). Increased exposure to green light resulted in increased GFP expression. These correlates to increased CcaS/R activity and MetE expression.

**Note:** GFP is used as a proxy for MetE expression because they are both under a CcaR promoter in all characterization experiments.

 ![](https://static.igem.org/mediawiki/parts/e/e3/Waterloo-GFP-Graph-Dec-7.png)

### Limitations of Our System

When comparing doubling times of replicates done on different days a significant amount of variation is seen between samples. However, when comparing replicates on the same day they match well.

 ![](https://static.igem.org/mediawiki/parts/3/30/Variation-dec7.png)
 ![](https://static.igem.org/mediawiki/parts/8/84/Chart-dec7.png)

**Solution**
In order to compensate for this day to day variation we will have to implement our Specific Model Predictive Controller. This model uses an optimization technique known as Moving Horizon Estimation (MHE). The control algorithm will take into account the day-to-day variability and adjust accordingly in order to still perform optimally.

## Waterloo iGEM 2018

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© 2018 Waterloo iGEM