iGEM 2017 — Team York

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# Team:York/Notebook

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[YORK](//2017.igem.org/Team%3AYork)

* [HOME](//2017.igem.org/Team%3AYork)
* [PROJECT](////2017.igem.org/Team%3AYork/Description)

  + [DESCRIPTION](//2017.igem.org/Team%3AYork/Description)
  + [DESIGN](//2017.igem.org/Team%3AYork/Design)
  + [APPLIED DESIGN](//2017.igem.org/Team%3AYork/Applied_Design)
  + [RESULTS](//2017.igem.org/Team%3AYork/Results)
  + [BACKGROUND](//2017.igem.org/Team%3AYork/Background)
  + [DOWNLOADS](//2017.igem.org/Team%3AYork/Downloads)
  + [CONTRIBUTION](//2017.igem.org/Team%3AYork/Contribution)
* [DIHM](https://2017.igem.org/Team%3AYork/Hardware)

  + [HARDWARE](https://2017.igem.org/Team%3AYork/Hardware)+ [SOFTWARE](https://2017.igem.org/Team%3AYork/Software)
    + [MEASUREMENT](https://2017.igem.org/Team%3AYork/Measurement)
* [COMMUNITY](https://2017.igem.org/Team%3AYork/Human_Practices)

  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AYork/Human_Practices)
  + [HP SILVER](https://2017.igem.org/Team%3AYork/HP/Silver)
  + [HP GOLD INTEGRATED](https://2017.igem.org/Team%3AYork/HP/Gold_Integrated)
  + [COLLABORATIONS](https://2017.igem.org/Team%3AYork/Collaborations)
* [OUR LAB](https://2017.igem.org/Team%3AYork/Achievements)

  + [NOTEBOOK](https://2017.igem.org/Team%3AYork/Notebook)
  + [PROTOCOLS](https://2017.igem.org/Team%3AYork/Experiments)
  + [SAFETY](https://2017.igem.org/Team%3AYork/Safety)
  + [DEMONSTRATE](https://2017.igem.org/Team%3AYork/Demonstrate)
  + [ACHIEVEMENTS](https://2017.igem.org/Team%3AYork/Achievements)
* [ABOUT US](https://2017.igem.org/Team%3AYork/Team)

  + [MEET THE TEAM](https://2017.igem.org/Team%3AYork/Team)
  + [ATTRIBUTION](https://2017.igem.org/Team%3AYork/Attributions)
  + [SPONSORS](https://2017.igem.org/Team%3AYork/Sponsors)

>

# Notebook

# Tracking Our Progress

## Notebook

### Week 1 (19 June - 25 June)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)
* ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

  #### Microscopy Division

  Had the first meeting of the division. Discussed timeline and the components we have available. For now, we will order only camera. Figured out that Biology Labs do not have any lasers and optical benches.

### Week 2 (26 June - 2 July)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  In the end of a last weeek, we decided to work with algae and bacteria. Biology Lab Safety talk for all team members. Prepared LB media for *E.coli* and pored it into plates. Freezig *E.coli* cells for an emergency. Checking plates for algae and liquid culture for transformation. Prepared for plasmid extraction via electrophoresis.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We were given access to Physics Labs. It will be our home for the next weeks. We watched laser safety video and started taking pictures with freshly arrived Raspberry Pi V2 Camera. Made prototype of the DIHM. How easy was that?!

### Week 3 (3 July - 9 July)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Digested and extracted pLM005. Prepared TAP media plates for algae. Performed colony PCR for ACA3.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Flower shape leaves pictures were obtained by shining laser on camera. Problem might be in IR filter on the lens of the camera. We ordered Raspberry Pi V2 NoIR camera which does not have IR filter. While waiting for a new camera, we were trying to make Rapsberry Pi to sent pictures automatically to desktop computer.

### Week 4 (10 July - 16 July)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Nanodroped and extracted PCR products. Re-streaked algae. Did cell counts through Optical Density and took pictures of Chlamydomonas. Currently we are devising growth experiments for algae and bacteria.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Received NoIR camera. Leaves shape pattern solved. We were trying to observe diffraction from single slit experiment with a laser. Intensity of a laser was lowered with polarisation filter to avoid overexposure of camera. We used converging lens available in the lab to focus diffraction pattern on CMOS. Unfortunately, it was hard to setup to capture clear image. We are thinking to acquire lens with small focal distance to make setup as compact as possible.

### Week 5 (17 July - 23 July)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Obtaied bacteria growth curve in 96 well plate. Ethanol toxicity growth test should be run next week for *E.coli*. Some new plates were made. Performed Alkaline lysis,

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Removed lens from camera which was done easily. Now, light is shinning directly on CMOS. Still we have problems with alignment of laser, slits and camera. Moreover, single slit was too bright for naked CMOS. Now, we are using double slit experiment to observe interference in best tradition of Thomas Young.

### Week 6 (24 July - 30 July)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Growth tests are continuing. We are getting curve for bacteria. Repeating PCR using Polymerase Q5 for MEX1.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    After imaging double slit interference pattern we are moving to object imaging comperable with microorganisms size - microbeads. In parallel, we are translating software code for image reconstruction from LabVIEW to MATLAB. We received laser from TU Darmstadt team which is iterested in building DIHM, too.

### Week 7 (31 July - 6 August)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Started *E.coli* transformation. Designing growth test with 24 well plate for algae. 96 wels plate well's are too small for algae. Set-up co-culture with untransformed microorganisms.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We were able to see single microbeads with our setup. Now, we have to be technical and measure precisely distances need for fully working DIHM. Also, we met with Dr Wilson to discuss our progress so far.

### Week 8 (7 August - 13 August)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Performed PCR for MEX1, again. Experimenting on our 'frankenstein' media for a co-culture. Co-culture testing in traditional media.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Looks like we will have to make image analysis in Python for a beginning. We still see the noise on captured images.

### Week 9 (14 August - 20 August)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Obtained algae growth curve. Next is algae ethanol tolerance test. Still adjusting new media for co-culture.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Last week we ordered a new laser and quality of the image changed drastically. Laser that we were using in the lab was inappropriate for Digital Holography as we have figured that out.

### Week 10 (21 August - 27 August)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Finally, we start transforming algae! In parallel, we run algae-bacteria depedence tests to find relationship between them.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Image recostruction software is almost ready. We are thinking about the design of DIHM and seeking for inspiration around the campus. Teflon mold was made for us this week which we will use to cast PDMS.

### Week 11 (28 August - 3 September)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  We have run initial transformation screening adn identified individual colonies containing assembled constructs.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We find difficult to attach PDMS chamber to a glass slide. We met with plasma researcher in your university to discuss plasma bonding options. Whereas, UI for image capturing and analysis is rapidly developed.

### Week 12 (4 September - 10 September)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Ordered BioBrick for *E.coli* that increases it's motility. Transformation did not proceed correctly.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We abandoned idea of using PDMS as a material for imaging chamber. Aluminium case for our microscope is ready. We hope to get all parts by next week.

### Week 13 (11 September - 17 September)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  It look like we will not be able to fix problem with algae transformation. It was ambitious task which was not possible to be done in such short period of time for us, but we still have the microscope.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Solution for imaging chamber was found by using acryl sheets and laser cutting. Desktop does not want to communicate with Raspberry Pi, but we will reconcile them.

### Week 14 (18 September - 24 September)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Offered sacrifice sample of algae and bacteria to Microscopy Division. Analysing data from growth tests for algae and bacteria.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We have imaged *Chlamydomonas reinhardtii* and *E.coli* separately and together. We figured out that millichamber we have manufactured are too thick for your laser. Instead, we are using glass slide and coverslip. UI is completed.

### Week 15 (25 September - 1 October)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Received BioBrick from iGEM HQ. We will transform *E.coli* to copmare motility of wild type and engineered organism.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    We compared results from last week imaging and automated cell counter. It looks like our setup is working and we are able to calculate concetration of the microorganisms. We will do imaging of yeast and other bacteria and algae.

### Week 16 (2 October - 8 October)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Transformed *E.coli*. If algae would be transformable so easily.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Ran high motility bacteria on your DIHM.

### Week 17 (9 October - 15 October)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Clenaing our lab space and helping to upload information to wiki.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Not much progress was done on laptop-microscope connection.

### Week 18 (16 October - 22 October)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Still clearing up the lab.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    All content is being uploaded to wiki.

### Week 19 (23 October - 29 October)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  Finished analysis of a growth curves and we are fully moved out of the lab. We will miss it.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Unfortunately, UI does not like computers other than were it was created.

### Week 19.5 (29 October - 1 November)

---

* ![](https://static.igem.org/mediawiki/2017/e/e9/IGEMYork_BiologyLogo.png)

  #### Biology Division

  We are working on wiki, poster and presentation preparation.

  * ![](https://static.igem.org/mediawiki/2017/3/36/IGEMYork_MicroscopeLogo.png)

    #### Microscopy Division

    Everybody is working hard on wiki and will do so until the deadline.

* ABOUT US
* We are an overgraduate team representing the University of York.
* Our Quicker Way to Analyse Co-Cultures (QWACC) is our entry into the Hardware track for iGEM 2017.

* LOCATION
* Department of Biology
* [University of York](//www.york.ac.uk)
* Heslington
* North Yorkshire
* YO10 5DD

* CONTACT US
* Email: igemyork@gmail.com
* You can also reach us through our social media via the links below.
* [![](//2017.igem.org/wiki/images/0/0c/Icon-facebook.png)](//www.facebook.com/igemyork/)
  [![](//2017.igem.org/wiki/images/6/6f/Icon-twitter.png)](//twitter.com/igemyork?lang=en)
  [![](//2017.igem.org/wiki/images/5/51/Icon-instagram.png)](//www.instagram.com/igemyork/)

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[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:York/Experiments

[YORK](//2017.igem.org/Team%3AYork)

* [HOME](//2017.igem.org/Team%3AYork)
* [PROJECT](////2017.igem.org/Team%3AYork/Description)

  + [DESCRIPTION](//2017.igem.org/Team%3AYork/Description)
  + [DESIGN](//2017.igem.org/Team%3AYork/Design)
  + [APPLIED DESIGN](//2017.igem.org/Team%3AYork/Applied_Design)
  + [RESULTS](//2017.igem.org/Team%3AYork/Results)
  + [BACKGROUND](//2017.igem.org/Team%3AYork/Background)
  + [DOWNLOADS](//2017.igem.org/Team%3AYork/Downloads)
  + [CONTRIBUTION](//2017.igem.org/Team%3AYork/Contribution)
* [DIHM](https://2017.igem.org/Team%3AYork/Hardware)

  + [HARDWARE](https://2017.igem.org/Team%3AYork/Hardware)+ [SOFTWARE](https://2017.igem.org/Team%3AYork/Software)
    + [MEASUREMENT](https://2017.igem.org/Team%3AYork/Measurement)
* [COMMUNITY](https://2017.igem.org/Team%3AYork/Human_Practices)

  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AYork/Human_Practices)
  + [HP SILVER](https://2017.igem.org/Team%3AYork/HP/Silver)
  + [HP GOLD INTEGRATED](https://2017.igem.org/Team%3AYork/HP/Gold_Integrated)
  + [COLLABORATIONS](https://2017.igem.org/Team%3AYork/Collaborations)
* [OUR LAB](https://2017.igem.org/Team%3AYork/Achievements)

  + [NOTEBOOK](https://2017.igem.org/Team%3AYork/Notebook)
  + [PROTOCOLS](https://2017.igem.org/Team%3AYork/Experiments)
  + [SAFETY](https://2017.igem.org/Team%3AYork/Safety)
  + [DEMONSTRATE](https://2017.igem.org/Team%3AYork/Demonstrate)
  + [ACHIEVEMENTS](https://2017.igem.org/Team%3AYork/Achievements)
* [ABOUT US](https://2017.igem.org/Team%3AYork/Team)

  + [MEET THE TEAM](https://2017.igem.org/Team%3AYork/Team)
  + [ATTRIBUTION](https://2017.igem.org/Team%3AYork/Attributions)
  + [SPONSORS](https://2017.igem.org/Team%3AYork/Sponsors)

# Protocols

# QWACC Team Methods

**In the interest of reproducibility, we would like to use this page to share the protocols that we have used during this project.
This library of protocols has been developed alongside our supervisors, to whom we are very grateful.
Please find, below, the exact methods we've used to generate our data and results.**

### **Growth Testing**

#### [*E. coli* LW06 Maltose Growth](#P2-collapse)

***E. coli* LW06 Medium Growth Test - Maltose**

**Tris phosphate and maltose concentrations: To determine how LW06 would grow on carbon source maltose (proposed export of *Chlamydomonas reinhardtii*). Performed in 96 well plates over 24 hours in 96 plate reader. OD reading taken every 30 minutes.**

1. **Grow stock culture of LW06 16-24 hours.**
2. **Dilute stock culture to produce inoculum with consistent cell density:**

1. Produce inoculum of between 0.5-1.0 OD.
2. Spin down culture in centrifuge (2500 rpm 2 minutes).
3. Remove supernatant and suspend in TP medium, repeat twice. (Do not do this for LB control culture.)

3. **Produce TP maltose stock solution:**

1. Make 10 g/L (29.21414 mM) maltose monohydrate solution.

1. 50 mL distilled water.
2. 0.5 g maltose.

4. **Make media dilutions in 96 well plates:**

1. Columns 1 & 3 add 200 μL of LB. Columns 2 & 4 add 200 μL of TP. Columns 1 & 2 and columns 3 & 4 will be blanks and growth controls, respectively.
2. Column 5 add 20 μL of TP.Maltose stock and 180 μL of TP.
3. Repeat for column 6-12 while increasing TP.Maltose volume by 20 μL for each column. Dilution method results in a 1-8 g/L gradient in the wells 5-12, respectively.

5. **Inoculate plate:**

1. 10 μL of LW06 to columns 3 to 12.

6. **Measure optical density of cultures:**

1. 96 well plate reader, run time 24 hours. One reading per 30 minutes. 48 total readings.

#### [Co-culture Ratio Experiment](#P3-collapse)

**Co-culture ratio experiment**

**Co-culture experiment: The growth disparity between *C. reinhardtii* and the *E. coli* LW06 strain is quite large. LW06 doubles every 28 minutes at 37°C while *C. reinhardtii* doubles once every 8 hours at 25°C, around 17 times slower than LW06. Considering this, we endeavoured to understand which ratios of initial inoculation would be required to ensure that *E. coli* does not completely saturate the culture.**

1. **Grow stock culture of LW06 16-24 hours.**
2. **Dilute stock culture to produce inoculum with consistent cell density:**

1. Produce inoculum of between 0.5-1.0 OD.
2. Spin down culture in centrifuge (2500 rpm 2 minutes).
3. Remove supernatant and suspend in TP media, repeat washing step twice.

3. **Grow stock culture of *C. reinhardtii* 7 days minimum (need long term stock culture from which to draw):**

1. Produce inoculum of 10 mL of between 0.5-1.0 OD.
2. Spin culture down in centrifuge (2500 rpm 2 minutes.
3. Remove supernatant and suspend in TP, repeat washing step twice.

4. **Produce TP maltose and TAP maltose stock media:**

1. Make 10 g/L (29.21414 mM) of TP.Maltose:

1. 50 mL TP in each tube.
2. 0.50 g maltose in each tube.

2. Make 10 g/L (29.21414 mM) of TAP.Maltose:

1. 50 mL TAP in each tube.
2. 0.50 g maltose in each tube.

5. **Fill 8 falcon tubes (50 mL) in the following manner:**

1. Tube 1 TP 25 mL.
2. Tube 2 TP 25 mL.
3. Tube 3 TAP 25 mL.
4. Tube 4 TAP 25 mL.
5. Tube 5 TP.M 25 mL.
6. Tube 6 TP.M 25 mL.
7. Tube 7 TAP.M 25 mL.
8. Tube 8 TAP.M 25 mL.

6. **Inoculate all tubes with 100 μL of *C. reinhardtii*. Inoculate the first tube in each pair with 10 μL and the second tube with 100 μL of LW06.**
7. **Leave to grow for 8 days in growth room at 25°C (we used an 8 hour day light cycle in the room).**
8. **Measure cell types at the end of 8 days via the following methods:**

1. *C. reinhardtii* haemocytometer microscope counting at a 104 dilution.
2. *E. coli* LW06 LB spread plating at a 104 dilution.

#### [*E. coli* LW06 Hblend Growth Test](#P4-collapse)

**LW06 Medium Growth Test - Hblend**

**Hblend (Home blend media) experiment: TP media is quite depleted in most micronutrients required for *E. coli* growth, as it is a media formulated for C.reinhardtii growth under photosynthetic growth. So, to try and solve this problem we decided to create a growth medium that could allow for the improved growth of LW06 and *C. reinhardtii*. Hblend media like M9 medium will act as a micronutrient supply with a variable carbon source. This test was performed in 96 well plates over 24 hours in 96 plate reader with an OD reading taken every 30 minutes.**

1. **Grow stock culture of LW06 16-24 hours.**
2. **Dilute stock culture to produce inoculum with consistent cell density:**

1. Produce inoculum of between 0.5-1.0 OD.
2. Spin down culture in centrifuge (2500 rpm 2 minutes).
3. Remove supernatant and suspend in TP and Hblend media, repeat twice. (Do not do this for LB control cuture.)

3. **Produce Home blend medium stock (500 mL):**

1. Prepare micronutrients:

1. 1 M NaCl: 4.28 mL
2. 1 M Na2HPO4: 23.875 mL
3. 1 M KH2PO4: 11.02 mL
4. 1 M NH4Cl: 9.345 mL
5. 0.1 M CaCl2: 1.7 mL
6. 1 M MgSO4: 1 mL
7. 28.5 μM (NH4)6Mo7O24: 0.5 mL
8. 0.1 mM Na2SeO3: 0.5 mL
9. 2.5 mM Zn x EDTA: 0.5 mL
10. 6 mM Mn x EDTA: 0.5 mL
11. 20 mM Fe x EDTA: 0.5 mL
12. 2 mM Cu x EDTA: 0.5 mL
13. Phosphate solution: 0.1875 mL
14. 2M Tris/acetate or tris/HCl, pH 7.4: 5 mL

2. Add distilled water 440.6 mL to make 500 mL.
3. Autoclave to sterilize.
4. Add 20 mL 20% glucose solution or other carbon source.

4. **Set up 96 well plates:**

1. Columns 1-6 add 200 μL of LB, TP.Maltose, TP.Glucose, Hblend, Hblend.Glucose and Hblend.Maltose, respectively.
2. Columns 7-12 add 190 μL of LB, TP.Maltose, TP.Glucose, Hblend, Hblend.Glucose and Hblend.Maltose, respectively.

5. **Inoculate plate:**

1. 10 μL of LW06 to each of columns 7-12 respective to washed down inoculating culture.

6. **Measure optical density of cultures:**

1. 96 well plate reader, run time 24 hours. One reading per 30 minutes, 48 readings total.

#### [*C. reinhardtii* Ethanol Tolerance](#P5-collapse)

***C. reinhardtii* Ethanol Tolerance**

**Experiment: The key to a self-sustaining (or even partially self-sustained) biological production culture is that the carbon provider (*C. reinhardtii*) is not killed by the product (ethanol) produced by the other organism (LW06) in the culture. So, we set up an ethanol tolerance test for *C. reinhardtii* in a 24 well plate.**

1. **Grow stock culture of *C. reinhardtii* 7 days minimum (need long term stock culture from which to draw):**

1. Produce inoculum of 10ml of between 0.5-1.0 OD.
2. Spin culture down in centrifuge (2000 rpm 2 minutes).
3. Remove supernatant and suspend in TP, repeat washing step twice.

2. **Make media dilutions in 24 well plates: one plate for TP medium, one for TAP medium.**

1. Plate 1 TAP:

1. Rows A-C TAP.
2. Row D distilled water.
3. Column 1 leave un-inoculated for all rows. A-C 1000 μL TAP; D 1000 μL distilled water.
4. Column 2 rows A-C 1000 μL TAP; row D 1000 μL distilled water. Inoculate with 250 μL of *C. reinhardtii*.
5. Column 3 rows A-C 990 μL TAP, 10 μL 100% ethanol; row D 990 μL distilled water, 10 μL 100% ethanol.
6. Repeat above for columns 4-6, increasing volume of ethanol and decreasing TAP/distilled water volumes in increments of 10 μL.
7. Ethanol gradient increases from 1% to 4% from column 3 to column 6, respectively.

2. Plate 2 TP:

1. Repeat above method, replacing TAP with TP.

3. **Inoculate plate:**

1. 250 μL of *C. reinhardtii* inoculum in columns 2-6.

4. **Measure optical density of cultures:**

1. 24 well plate reader (λ = 720 nm). Readings taken once ~24 hours. When not being measured, keep in 25°C incubator room/cabinet with 8 hour day cycle.

### **DIHM & Milli-fluidic Device Methodology**

#### [Milli-fluidic Chamber Casting from PDMS](#PDMS-collapse)

**Milli-fluidic Chamber Casting from PDMS**

**Crating chamber for imaging microorganisms in liquid phase using transparent non-toxic polymer PDMS.**

1. **Gather materials:**

1. PDMS
2. PDMS curing agent
3. Falcon tube 20 ml
4. Iso-propyl alcohol
5. Magic tape
6. Spatula
7. Milli-fluidic Chamber mould
8. Pipettes

2. **Clean mould**

1. Rinse mould with Iso-propyl alcohol and then with deionised water. Left drying.
2. If there some pieces of rubbish on the mould use magic tape to remove them.

3. **Curing PDMS**

1. Pour 4 ml of PDMS and 0.4 ml of curing agent into falcon tube and mix well with disposable stick or metallic spatula.
2. Put falcon tube with PDMS into centrifuge. Use another falcon tube with water as counterweight. Spin for 2 min at 2500 rpm.

4. **Moulding**

1. Take falcon tube with PDMS out of centrifuge. It should not contain any bubbles. Pour it into the mould.
2. Put mould with PDMS into the oven at 80 Centigrade for 2 hours. If it is not enough, leave in oven for longer until PDMS fully solidifies.
3. Take mould with PDMS out of the oven and let it cool for a minute. Take chamber out with spatula.

#### [Milli-fluidic Chamber Production](#Polyacryl-collapse)

**Milli-fluidic Chamber Production**

**Low cost chamber for imaging microorganisms**

1. **Gather materials:**

1. Acrylic sheet
2. Transparent tubing d = 1.5 mm
3. Epoxy glue
4. Glass slide and coverslip
5. Blu tack

2. **Design**

1. Chamber design will depend on what type of microorganisms you are working and imaging technique you are using. Tubing has to be taken into account as well.
2. Main parameters for our chamber:
   * Height should be 2 mm - 5 mm. Physical limitation of laser we are using.
   * We want channe to be l as narrow as possible. It will match diameter of tubing.
   * No sharp edges. This will prevent algae from sticking in chamber.
   * Cheap and easy to produce.

3. **Drafting**

1. Draw your chamber on paper and then transfer it into 3D CAD specifying all dimensions. We were using Fusion 360. It is free for students.
2. Engraving for tubing is a tricky part. It is unique for every laser/material combination. You will have to play around with settings to achieve deepness that you want.
3. All laser cutting was done by a trained specialist.
4. It is important to have 3D model of the chamber. All machines are different and do not support same type of files, but having one file that illustrates your design in detail will help to transfer it onto laser cutter.

4. **Manufacturing**

1. Laser cut chamber from acrylic sheet.
2. It will take multiple attempts to make right engraving.

5. **Tubing**

1. Cut enough tubing for peristaltic pump. Two tubes 30 cm long should be enough.
2. Apply super or epoxy glue around one end of each tube and put it into engraved part of the chamber. If necessary, add some glue from sides were tubing sticks out.
3. Put glued chamber into oven to accelerate process. 4 hours at 60 Centigrade is enough for epoxy glue.

6. **Assembly**

1. Most innovative part of the project.In order to stick glass slide and coverslip to chamber we are using blu tack.
2. Create thin rolls around 0.5 mm in diameter out of blu tack.
3. Make an outline around channel from both sides.
4. Squeeze chamber between slide and cover slip.

#### [Diffraction Pattern Image Capturing (Initial Testing)](#P6-collapse)

**Diffraction Pattern Imaging with Raspberry Pi**

**Imaging diffraction patterns cheaply: in order to keep the DIHM cheap, we planned to use a Raspberry Pi 3 and Camera Module V2 as the image capturing device. We tested its capabilities on an optical bench.**

1. **Gather materials:**

1. Raspberry Pi 3 and power supply.
2. Camera Module V2 (remove lens to expose CMOS).
3. HDMI cable.
4. USB keyboard.
5. USB mouse.
6. Monitor (with HDMI input).
7. Optical bench (and clamps, stands, related accessories).
8. Laser (we used a 635 nm laser diode with collimating lens at this stage, though the collimating lens was omitted in later tests due to its projection of concentric rings into images).
9. Glass slides and cover slips.
10. Microbeads of known diameter.

2. **Attach Camera Module V2 to Raspberry Pi.**
3. **Set up Raspberry Pi 3 for ease of control:**

1. Connect HDMI from Pi to monitor.
2. Connect mouse and keyboard to the Pi.
3. Connect power supply to Pi and switch on Pi and monitor.

4. **Place the Camera Module and laser at opposite ends of the optical bench.**
5. **Align the laser with the Camera Module's CMOS:**

1. Place a piece of card in front of the laser, in a clamp, on the optical bench.
2. Make a mark where the laser meets the card.
3. Move the card along the optical bench, towards the camera.
4. Adjust the laser and repeat until it follows the mark on the card all the way along the bench.
5. Ensure that the Camera Module is placed directly in the path of the laser now that it is aligned with the bench.

6. **Add a suitably diluted (trial and error) sample of microbeads to a slide.**
7. **Place the slide (in a clamp) on the optical bench, as close to the CMOS as possible.**
8. **Take images of the microbeads' diffraction pattern:**

1. Open the terminal (command prompt) on the Pi's interface.
2. Use the raspistill command to capture a monochrome image:

* raspistill -e png -cfx 128:128 -w 512 -h 512 -o /home/pi/ExampleFilename.png
* *The above exemplar code takes a monochrome, 512 x 512 pixel image via the Camera Module and saves the picture in PNG format in the /home/pi/ folder under the filename "ExampleFilename.png".*
* Taking pictures in monochrome prevents the Pi from trying to colour balance the images, which can alter the clarity, while keeping it to 512 x 512 pixels allows for quick data processing in later stages. PNG format is used as it is lossless, preserving all quality.

9. **Circular diffraction patterns, caused by the microbeads, should be apparent in the captured picture.**

#### [Organism Image Capturing (Glass Slides + Mouse and Keyboard)](#P7-collapse)

**Organism Image Capturing on Glass Slides, Mouse and Keyboard**

**Taking images for hologram production, using glass slides and a mouse and keyboard. Our original plan, to use a flow chamber to cycle through liquid cultures/co-cultures in a closed system, needed improvement. Hence, we resorted to using glass slides. Further, our control software had bugs that prevented us from using it, so we used a mouse, keyboard and python programs to manually capture images.**

**At least 20 images are required to make an appropriate background image from which holograms can be created, so we tried to take between 20 and 50 images of each sample. We have written software to make this easier (see Downloads: "Taking Series of Images (Python)"). This protocol assumes that it is stored on the Pi from the beginning.**

1. **Gather materials:**

1. Constructed DIHM (as per the [Hardware](//2017.igem.org/Team%3AYork/Hardware) page).
2. Organism of choice.
3. TAP medium.
4. HDMI cable.
5. USB keyboard.
6. USB mouse.
7. Monitor (with HDMI input).
8. Glass slides and cover slips.
9. Size 4 Allen key.
10. Small spirit level(s).

2. **Arrange platforms for desired magnification:**

1. Use Allen key to loosen/tighten into place.
2. Use spirit level to ensure lens platform is level with base of DIHM.

* *For our best results, we used a sample to lens distance of around 1.2 cm (placing slides directly on top of lens platform) and a lens to camera distance of 6 cm, giving a magnification of 5x.*

3. **Set up Pi connections:**

1. Connect HDMI, USB mouse and USB keyboard to Pi through base of DIHM.
2. Connect HDMI to monitor, also.
3. Plug in Pi's power supply to mains voltage and switch on. (Laser will become active.)

4. **Prepare 10-12 µL of suitably TAP-diluted (trial and error) sample of the organism on a glass slide.**
5. **Place the slide at the desired height on the DIHM.**
6. **Take images:**

1. Begin moving the slide across the camera's field of view, slowly.
2. Open the terminal (command prompt) on the Pi's interface.
3. While moving the slide, use the python program to capture 50 images:

* python /home/pi/Take50Images.py
* *Try to avoid passing the edges of the cover slip while taking images, as these pictures will be averaged to create a background. This can then be used to create holograms.*

7. **50 images should now be saved in the folder "/home/pi/DATE--ORGANISM--DILUTION--MAGNIFICATION/".**

#### [Organism Image Capturing (Milli-fluidic Chamber + Mouse and Keyboard)](#P8-collapse)

**Organism Image Capturing with a Milli-fluidic Chamber, Mouse and Keyboard**

**Taking images for hologram production, using a milli-fluidic chamber and a mouse and keyboard. We connected the chamber to liquid culture in a closed system. Our control software had bugs that prevented us from using it, so we used a mouse, keyboard and python programs to manually capture images.**

**At least 20 images are required to make an appropriate background image from which holograms can be created, so we tried to take between 20 and 50 images of each sample. We have written software to make this easier (see Downloads: "Taking Series of Images (Python)"). This protocol assumes that it is stored on the Pi from the beginning.**

1. **Gather materials:**

1. Constructed DIHM (as per the [Hardware](//2017.igem.org/Team%3AYork/Hardware) page).
2. Constructed milli-fluidic chamber (as per protocol).
3. Peristaltic pump with flow-rate on order millilitres.
4. Organism of choice in liquid culture.
5. TAP medium.
6. 100% ethanol.
7. HDMI cable.
8. USB keyboard.
9. USB mouse.
10. Monitor (with HDMI input).
11. Size 4 Allen key.
12. Small spirit level(s).

2. **Arrange platforms for desired magnification:**

1. Use Allen key to loosen/tighten into place.
2. Use spirit level to ensure lens platform is level with base of DIHM.

* *For our best results, we used a sample to lens distance of around 1.2 cm (placing slides directly on top of lens platform) and a lens to camera distance of 6 cm, giving a magnification of 5x.*
* *However, when we used the milli-fluidic chambers, we hadn't yet built the lens platform. So, we placed the chambers directly over the Pi's Camera Module, giving an effective magnification of 1x.*

3. **Set up Pi connections:**

1. Connect HDMI, USB mouse and USB keyboard to Pi through base of DIHM.
2. Connect HDMI to monitor, also.
3. Plug in Pi's power supply to mains voltage and switch on. (Laser will become active.)

4. **Clean the milli-fluidic chamber:**

1. Pump 100% ethanol through the chamber to sterilise it.
2. Pump TAP medium through the chamber to ensure that no ethanol remains.

5. **Connect the chamber and liquid culture to the pump.**
6. **Place the chamber at the desired height on the DIHM.**
7. **Take images:**

1. Switch on the pump.
2. Open the terminal (command prompt) on the Pi's interface.
3. Ensure the chamber is in the camera's field of view, then use the python program to capture 50 images:

* python /home/pi/Take50Images.py

8. **50 images should now be saved in the folder "/home/pi/DATE--ORGANISM--DILUTION--MAGNIFICATION/".**

#### [Organism Image Capturing (Milli-fluidic Chamber + Control Software)](#P9-collapse)

**Organism Image Capturing with a Milli-fluidic Chamber and Control Sotware**

**Taking images for hologram production, using a milli-fluidic chamber and control software. We connected the chamber to liquid culture in a closed system. Our control software had bugs that prevented us from using it during the testing phase, so we have written a protocol detailing how we would have used it, had we been able to.**

1. **Gather materials:**

1. Constructed DIHM (as per the [Hardware](//2017.igem.org/Team%3AYork/Hardware) page).
2. Constructed milli-fluidic chamber (as per protocol).
3. Peristaltic pump with flow-rate on order millilitres.
4. Organism of choice in suitably diluted liquid culture.
5. TAP medium.
6. 100% ethanol.
7. Computer capable of running control software.
8. Ethernet cable.
9. Size 4 Allen key.
10. Small spirit level(s).

2. **Arrange platforms for desired magnification:**

1. Use Allen key to loosen/tighten into place.
2. Use spirit level to ensure lens platform is level with base of DIHM.

3. **Set up Pi connections:**

1. Connect ethernet cable from control computer to Pi through base of DIHM.
2. Plug in Pi's power supply to mains voltage and switch on. (Laser will become active.)

4. **Clean the milli-fluidic chamber:**

1. Pump 100% ethanol through the chamber to sterilise it.
2. Pump TAP medium through the chamber to ensure that no ethanol remains.

5. **Connect the chamber and liquid culture to the pump.**
6. **Place the chamber at the desired height on the DIHM.**
7. **Take images using the control software:**

1. Turn on the pump.
2. Ensure the chamber is in the camera's field of view.
3. Run the control program.
4. Follow the instructions given within the software.

*We intended for the control software to operate as follows:*

*The control software takes a series of images at regular time intervals, while the culture is pumped through the chamber. It then renders one of the images into a hologram and detects the number of cells of a given size.*

### **DNA Imaging, Analysis and Quantification**

#### [1% Agarose Gel for Electrophoresis](#P10-collapse)

**1% Agarose Gel for Electrophoresis**

1. **Gather materials:**

1. Agarose powder: 0.6 g
2. TAE (x1): 60 mL
3. SYBR Safe: 6 μL
4. Microwave safe flask
5. Tray for gel casting
6. Well comb

2. **Mix Agarose powder and TAE into microwaveable flask.**
3. **Microwave for 1-3 min until agarose fully dissolved:**

1. Microwave in 30s pulses, swirling the flask occasionally as the solution heats up.
2. **Do not overboil** the solution, as some of the buffer will evaporate.

4. **Let agarose solution cool down to about 50°C. (Around when you can comfortably keep your hand on the flask.) This should take about 5 minutes.**
5. **Add SYBR Safe.**
6. **Pour the agarose into a gel tray and place well comb.**

* *Different well combs should be used for qualitative or extraction gels.*

7. **Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 mins, until completely solidified.**

#### [Loading and Running Agarose Gel](#P11-collapse)

**Loading and Running Agarose Gel**

1. **Gather materials:**

1. 1% agarose gel
2. TAE buffer (x1)
3. Loading dye
4. Ladder
5. DNA samples/li>- PCR tubes
   - Electrophoresis unit
   - Voltage source

2. **Add DNA and loading dye in PCR tube and mix.**

* *We use NEB Purple (x6) gel loading dye.*

3. **Place gel in electrophoresis chamber.**
4. **Fill tank with TAE to submerge gel.**
5. **Load 6 μL of ladder and 6 μL of reactions.**

* *We use NEB Quick-Load Purple 2-Log DNA Ladder.*
* *Other ladders (specified in data analysis) were used when different band size distribution was required.*
* *For gel extraction larger sample amounts were loaded.*

6. **Run gel at 80 V until bands separated.**
7. **Image using UV box.**

* *Blue light transilluminator was used to image gels for extraction.*

#### [Nanodrop](#P12-collapse)

**Nanodrop**

1. **Add 1.2 µl of pure water to the pedestal and lower the arm.**
2. **Initialise the instrument and take blank measurement (as a reference).**
3. **Load 1.2 µl of the plasmid prep onto the pedestal.**
4. **Load 6 μL of ladder and 6 μL of reactions.**
5. **Close the sampling arm and press “Measure” on the computer screen.**
6. **Make a note of:**

* *The concentration given (ng/µl).*
* *The ratio of 260/280 nm. (This should be around 1.9 if the DNA is pure).*

### **Media, Buffers and Solutions**

#### [Homeblend Medium Recipe](#P13-collapse)

**Homeblend Medium Recipe**

**This recipe is an attempt to create a supplemented TAP/TP to suit E. coli growth, as TAP is highly deficient on several elements.**

**The recipes for (NH4)6Mo7O24, Na2SeO3, Zn×EDTA, Mn×EDTA, Fe×EDTA, Cu×EDTA, Phosphate solution, tris-acetate, and tris-HCl can be found in the recipe for TAP/TP media.**

1. **Medium Recipe, 1 L**

   | Ingredient | Stock Concentration | Volume |
   | --- | --- | --- |
   | NaCl | 1 M | 8.56 mL |
   | Na2HPO4 | 1 M | 47.75 mL |
   | KH2PO4 | 1 M | 22.04 mL |
   | NH4Cl | 1 M | 18.69 mL |
   | CaCl2 | 0.1 M | 3.4 mL |
   | MgSO4 | 1 M | 2 mL |
   | (NH4)6Mo7O24 | 28.5 μM | 1 mL |
   | Na2SeO3 | 0.1 mM | 1 mL |
   | Zn x EDTA | 2.5 mM | 1 mL |
   | Mn x EDTA | 6 mM | 1 mL |
   | Fe x EDTA | 20 mM | 1 mL |
   | Cu x EDTA | 2 mM | 1 mL |
   | Phosphate solution |  | 0.375 mL |
   | Tris/acetate or tris/HCl, pH 7.4 | 2 M | 10 mL |
2. **Add distilled water to bring the volume up to 980 mL.**
3. **Autoclave to sterilise.**
4. **Add 20 mL 20% glucose solution, or other carbon source of your choice, to bring the solution up to 1 L.**

#### [M9 Recipes](#P14-collapse)

**M9 Stock Recipes**

1. 1. **M9 Salts, 1 L**

      | Ingredient | Mass |
      | --- | --- |
      | Na2HPO4.7H2O | 64 g |
      | KH2PO4 | 15 g |
      | NaCl | 2.5 g |
      | NH4Cl | 5.0 g |
   2. **Add distilled water to bring the volume up to 1 L.**
   3. **Autoclave to sterilise.**
2. 1. **M9 Medium, 1 L**

      | Ingredient | Volume |
      | --- | --- |
      | M9 Salts | 200 mL |
      | MgSO4, 1 M | 2 ml |
      | CaCl2, 1 M | 100 μl |
   2. **Add distilled water to bring the volume up to 980 mL.**
   3. **Autoclave to sterilise.**
   4. **Add 20 mL 20% glucose, or other carbon source of your choice.**

#### [TAP, TP and Stock Solutions Recipes](#P15-collapse)

**Stock Recipes**

1. 1. **TAP Salts, 100 mL**

      | Ingredient | Mass |
      | --- | --- |
      | CaCl2.2H2O | 0.2 g |
      | MgSO4.7H2O | 0.4 g | *For -S, use 3.3 g MgCl2.6H2O* |
      | NH4Cl | 1.5 g |
   2. **Add distilled water to bring the volume up to 100 mL.**
   3. **Autoclave to sterilise.**
2. 1. **Tris-acetate Solution, 2 M, 100 mL**

      | Ingredient | Amount |
      | --- | --- |
      | Trizma base | 24.2 g |
      | Glacial acetic acid | 10 ml |
   2. **Add distilled water to bring the volume up to 100 mL.**
   3. **Filter using 0.2 µm pore OR autoclave using liquid 60 cycle (at least 30 minutes to completely dissolve Tris base) to sterilise.**
   * *Note: to dissolve Trizma base in a timely manner, stir vigorously and heat gently. It will take some time.*
3. 1. **Tris-HCl Solution, 2 M, 100 mL**

      | Ingredient | Amount |
      | --- | --- |
      | Trizma base | 24.2 g |
   2. **Add around 50 mL distilled water, heat and stir to dissolve.**
   * *Note: DO NOT add too much water; a substantial amount of HCl will be added in the next step.*
   * *For the next step, ensure the use of a Tris appropriate pH probe, since some types of probe are not suitable.*3. **Adjust the pH of your solution using 5 M or higher HCl solution.**
   4. **Add distilled water to bring volume to 100 mL.**
   5. **Sterilize by filtering using 0.2 µm pore OR autoclave.**
4. 1. **Phosphate Solution, 100 mL**

      | Ingredient | Mass |
      | --- | --- |
      | K2HPO4 | 28.8 g |
      | KH2PO4 | 14.4 g |
   2. **Add distilled water to bring volume to 100 mL.**
   3. **Sterilize by filtering using 0.2 µm pore.**

**Trace Elements**

1. 1. **EDTA-Na2**
      1. **Make concentrated stock solution, 125 mM, 300 mL**

         | Ingredient | Mass |
         | --- | --- |
         | EDTA-Na2 | 13.959 g |

         * Add to 250 mL distilled water, titrate to pH 8.0 with trace element grade KOH, ~ 1.7 g.
         * Add distilled water to 300 mL.
      2. **Make diluted stock solution, 25 mM, 250 mL**
         * Add 50 mL of 125 mM concentrated stock to 200 mL distilled water.
2. 1. **(NH4)6Mo7O24**
      1. **Make concentrated stock solution, 285 μM, 250 mL**

         | Ingredient | Mass |
         | --- | --- |
         | (NH4)6Mo7O24 | 0.888 g |

         * Add distilled water to 250 mL.
      2. **Make diluted stock solution, 28.5 μM, 250 mL**
         * Add 25 mL of 280 μM concentrated stock to 225 mL distilled water.
3. 1. **Na2SeO3**
      1. **Make concentrated stock solution, 1 mM, 250 mL**

         | Ingredient | Mass |
         | --- | --- |
         | Na2SeO3 | 0.043 g |

         * Add distilled water to 250 mL.
      2. **Make diluted stock solution, 0.1 mM, 250 mL**
         * Add 25 mL of 1 mM concentrated stock to 225 mL distilled water.
4. 1. **Zn x EDTA**

      | Ingredient | Amount |
      | --- | --- |
      | ZnSO4.7H2O | 0.018 g |
      | EDTA-Na2, 125 mM | 12 mL |

      * Add distilled water to 250 mL.
5. 1. **Mn x EDTA**

      | Ingredient | Amount |
      | --- | --- |
      | MnCl2.4H2O | 0.297 g |
      | EDTA-Na2, 125 mM | 12 mL |

      * Add distilled water to 250 mL.
6. 1. **Fe x EDTA**

      | Ingredient | Amount |
      | --- | --- |
      | FeCl3.6H2O | 1.35 g |
      | EDTA-Na2 | 2.05 g |
      | Na2CO3 | 0.58 g |

      * Combine EDTA-Na2 and Na2CO3 in water, mix until dissolved.
      * Add FeCl3.6H2O.
      * Add distilled water to bring the volume up to 250 mL.
      + *Note: DO NOT use the EDTA-Na2 concentrate for this step.*
7. 1. **Cu x EDTA**

      | Ingredient | Amount |
      | --- | --- |
      | CuCl2.2H2O | 0.085 g |
      | EDTA-Na2, 125 mM | 4 mL |

      * Add distilled water to 250 mL.

**TAP Recipe**

1. 1. **TAP, 1 L**

      | Ingredient | Amount |
      | --- | --- |
      | Tris-acetate, 2 M | 10 mL |
      | TAP salts | 25 mL |
      | Phosphate solution | 0.375 mL |
      | EDTA-Na2, 25 mM | 1 mL |
      | (NH4)6Mo7O24, 28.5 µM | 1 mL |
      | Na2SeO3, 0.1 μM | 1 mL |
      | Zn x EDTA, 2.5 mM | 1 mL |
      | Mn x EDTA, 6 mM | 1 mL |
      | Fe x EDTA, 20 mM | 1 mL |
      | Cu x EDTA, 2mM | 1 mL |
      | Agar (for plates) | 15 g |
   2. **Add to 900 mL distilled water.**
   3. **Adjust to pH 7.4.**
   4. **Add distilled water to bring the volume up to 1 L.**
   5. **Autoclave to sterilise.**

**TP Recipe**

1. 1. **TP, 1 L**

      | Ingredient | Amount |
      | --- | --- |
      | Tris-HCl, 2 M | 10 mL |
      | TAP salts | 25 mL |
      | Phosphate solution | 0.375 mL |
      | EDTA-Na2, 25 mM | 1 mL |
      | (NH4)6Mo7O24, 28.5 µM | 1 mL |
      | Na2SeO3, 0.1 μM | 1 mL |
      | Zn x EDTA, 2.5 mM | 1 mL |
      | Mn x EDTA, 6 mM | 1 mL |
      | Fe x EDTA, 20 mM | 1 mL |
      | Cu x EDTA, 2mM | 1 mL |
      | Agar (for plates) | 15 g |
   2. **Add to 900 mL distilled water.**
   3. **Adjust to pH 7.4.**
   4. **Add distilled water to bring the volume up to 1 L.**
   5. **Autoclave to sterilise.**

#### [TAE Buffer](#P16-collapse)

**TAE Buffer**

1. **For 1 L of x50 stock:**

   | Ingredient | Amount |
   | --- | --- |
   | Tris-base | 242 g |
   | Acetate (100% acetic acid) | 57.1 mL |
   | EDTA (0.5 M sodium EDTA) | 100 mL |
2. **Add H2O up to 1 L.**

#### [LB Recipe](#P17-collapse)

**LB Medium**

1. **For 1 L:**

   | Ingredient | Amount |
   | --- | --- |
   | Tryptone | 10 g |
   | Yeast Extract | 5 g |
   | NaCl | 10 g |
   | For plates, Agar | 12 g |
2. **Add to 900 mL H2O**
3. **Adjust pH to 7.5 with NaOH**
4. **Adjust volume to 1 L with distilled water.**

### **Transformations and Preparation**

#### [*Chlamydomonas* Transformation](#P1-collapse)

***Chlamydomonas* Transformation**

1. **Preparing *Chlamydomonas* batch culture for transformation**

* Inoculation to produce a 4 L culture:

1. Inoculate 50 mL of TAP with a single, fresh colony from a TAP streak plate.

* *Use a 250 mL conical flask for efficient aeration and mixing while shaking.*

2. Grow, while continuously shaking, for 5 days at 50 – 100 μE until culture density reaches ~ 1 x 107 cells/mL.
3. Transfer 50 mL into 400 mL of fresh TAP.
4. Grow while shaking for another 2 days at 50 – 100 μE until culture density reaches ~ 8-9 x 106 cells/mL.
5. Inoculate 4L of fresh TAP with all 450 mL of *Chlamydomonas*, grow while bubbling air through and continuously stirring for ~30 hours at 100 μE until culture density reaches 2-4 x 106 cells/mL.

* *Ensure the final inoculation uses an exponential-phase culture.*
* *For smaller (up to 1 L) volumes, this step can be done in a conical flask with continuous shaking.*
* *The high volume is in case the culture does not reach the desired concentration, in which case a large volume of cells can be spun down for transformation.*

2. **Transformation**

* *Work with open cultures should be performed in an appropriate flow hood to decrease contamination risk.*
* *Use 50 mL Chlamydomonas, at 2-4 x 106 cells/mL, per transformation.*
* *Each transformation requires 5 x 107 cells.*

* **Materials to prepare**

1. TAP sucrose, 10 mM sucrose – 15 mL per transformation
2. Selection plates

+ *Plate preferences depend on the type of screening to be performed.*
+ *We used rectangular plates (250 mm x 250 mm).*
+ *1.5% TAP agar with appropriate antibiotic.*
+ *Label on the side to avoid interference with the colony picker camera during selection.*

3. DNA insert cassettes digested with appropriate enzyme
4. Electroporation cuvettes
5. Centrifuge tubes
6. Glass L-shaped spreaders
7. Pipettes and tips

* **Harvesting**

1. Aliquot *Chlamydomonas* culture into centrifuge tubes.
2. Pellet *Chlamydomonas* culture using centrifuge (4 minutes at 1000x g).
3. Discard supernatant and avoid disturbing cell pellet.

* **Plating and growth**

1. Use TAP 40 mM sucrose to dilute cell pellet to 2 x 108 cells/mL.
2. Add 250 μL of *Chlamydomonas* culture per electroporation cuvette.
3. Incubate at 16°C fo 5 minutes.
4. Prepare recovery tubes (15 mL falcon tubes) with 8 mL TAP sucrose.

+ *Steps v-vii should be performed as quickly as possible. Especially step vii.*
+ *Before electroporation, dry the cuvette.*

5. Add transformation cassette DNA (14.5 ng/kb cassette per 250 µL) and mix briefly.

+ *Do not mix by repeatedly pipetting. Use the pipette tip to mix by stirring.*

6. Electroporator should be set to 800 V, 25 μF.
7. Administer pulse and immediately add the mixture from the cuvette to the recovery tube.

* **Electroporation**

1. After all electroporations are complete, transfer recovery tubes to a shaker, and grow for 6 hours in the dark.
2. Centrifuge the recovery tubes for 4 min at 1000x g.
3. Discard most of the supernatant, leaving about 500 µL.
4. Resuspend the pellet in the residual supernatant and spread on a transformation plate using a glass spreader.
5. After all plates are adequately dry, place them upside-down in stacks in plastic bags, and return to growth room.
6. Incubate transformation plates at 2-10 µE for 1-2 days.
7. Increase the light intensity to 25-50 µE for 8-12 days.

+ *This should yield >2 mm colonies that can be used for selection.*

#### [Alkaline Lysis](#P18-collapse)

**Alkaline Lysis**

Stock Recipes

1. **Solution I**

   | Ingredient | Final Solution Concentration |
   | --- | --- |
   | Tris/HCl pH 8 | 25 mM |
   | EDTA | 10 mM |
   | Glucose | 50 mM |
2. **Solution II**

   | Ingredient | Final Solution Concentration |
   | --- | --- |
   | NaOH | 200 mM |
   | SDS | 1% |
3. **Solution III**

   | Ingredient | Final Solution Concentration |
   | --- | --- |
   | Potassium acetate | 3 M |

Protocol

1. Inoculate 5 mL LB (with antibiotic for selection) with a single colony at 37°C, shaking at 360 rpm, overnight.
2. Pellet 1.5 ml of the culture.
3. Resuspend the pellet in cold solution I, vortex to ensure the pellet resuspends.

* *Note: DO NOT vortex after step 3.*

4. Add 200ul solution II, invert gently twice, place on ice for 3 minutes, to lyse the cells.

* *Note: DO NOT keep on ice for more than 5 minutes.*

5. Add 150 µl solution III, mix by inverting tubes gently, put on ice.
6. Centrifuge in microfuge at full speed for 5 minutes, preferably at 3°C, to pellet cell membranes, etc.
7. Transfer supernatant to new tube, add 400 µl pheno/chloroform to supernatant, mix by inverting, then spin in microcentrifuge for 5 minutes, max speed, to separate the layers.
8. Transfer the upper phase to a new eppendorf add 600 µl 2-propanol, then leave on ice 10 minutes.
9. Spin at room temperature, 5-10 minutes, max speed, to pellet DNA and RNA. Dump the supernatant, rinse pellet in 200 µl ice cold 70% ethanol.
10. Centrifuge full speed 5 minutes, dump the supernatant, then leave tube open for 5 minutes to dry.
11. Add 50 µL of tris/HCl, 10 mM, pH 8 to resuspend pellet.

#### [Competent Cell Preparation](#P19-collapse)

**Competent Cell Preparation**

**This protocol was provided by the iGEM advisors at the University of York with the advice that it is important that everything is kept ice cold and sterile.**

1. **Inoculate 2 separate 250 mL flasks containing 50 mL of media with 500 µL of culture from an overnight.**
2. **Grow with aeration until OD 650 reaches 0.5-0.6 (approx. 3 hours).**
3. **Transfer the culture into sterile 50 mL Falcon tubes and chill on ice for 5 minutes.**
4. **Spin down at 5000 rpm for 10 minutes at 4°C.**
5. **Discard supernatant and resuspend the pellet in 10 mL ice cold sterile 0.1 M CaCl2**
6. **Chill on ice for 20 minutes.**
7. **Spin down at 5000 rpm for 10 minutes at 4°C.**
8. **Discard supernatant and resuspend in 1.4 mL of 0.1 M CaCl2 mixed with 0.6 mL of 50% glycerol (ice cold).**
9. **Aliquot 100 µL of culture into 1.5 mL sterile Eppendorfs.**
10. **Freeze at -80°C.**

#### [*E. coli* Single Tube Transformation](#P20-collapse)

***E. coli* Single Tube Transformation**

**This protocol was adapted from a protocol provided by iGEM.**

1. **Materials**

1. DNA to be transformed
2. 10 pg/µL Positive transformation control DNA
3. Competent Cells (50 µL per sample)
4. 1.5 mL Microtubes
5. SOC Media (950 µL per sample)
6. Petri plates with LB agar and antibiotic for selection
7. Ice & ice bucket
8. 42°C water bath
9. 37°C incubator
10. Sterile spreader or glass beads
11. Pipettes and tips
12. Microcentrifuge

2. **Procedure**

1. Label and pre-chill 1.5 mL tube.
2. Thaw competent cells on ice (10-15 minutes)
3. Pipette 50 µL of competent cells into 1.5 mL tube.
4. Pipette 1 µL of resuspended DNA into 1.5 mL tube:

1. Gently mix by pipetting.
2. Keep all tubes on ice.

5. Close 1.5 mL tubes, incubate on ice for 30 minutes:

1. Gently agitate by flicking but return to ice immediately.

6. Heat shock tubes at 42°C for 45 seconds.
7. Incubate on ice for 5 minutes.
8. Pipette 950 µl SOC media to each transformation:

1. SOC media should be warmed to room temperature before use.

9. Incubate at 37°C for 1 hour, shaking at 200-300 rpm.
10. Pipette 100 µL of each transformation onto petri plates - spread immediately.
11. Incubate transformations overnight (14-18 hours) at 37°C.
12. Pick and screen for transformed *E. coli*.

### **Miscellaneous**

#### [List of Manufacturers' Protocols](#P21-collapse)

**List of Manufacturers' Protocols**

1. **DNA extraction**

* QIAprep by QIAGEN
* [QIAprep](http://www.qiagen.com/resources/download.aspx?id=22df6325-9579-4aa0-819c-788f73d81a09&lang=en)

2. **Qualitative PCR**

* GoTaq® G2 DNA polymerase by Promega
* [GoTaq® G2 DNA polymerase](https://www.promega.co.uk/-/media/files/resources/protocols/product-information-sheets/g/gotaq-g2-dna-polymerase-protocol.pdf)

3. **Cloning PCR**

* Phusion High-Fidelity DNA Polymerase by Thermo Scientific
* [Phusion High-Fidelity DNA Polymerase](https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0012393_Phusion_HighFidelity_DNAPolymerase_UG.pdf)
* Q5® Hot Start High-Fidelity 2X Master Mix by NEB
* [Q5® Hot Start High-Fidelity 2X Master Mix](https://www.neb.com/protocols/2012/08/30/protocol-for-q5-hot-start-high-fidelity-2x-master-mix-m0494)

4. **PCR cleanup and gel extractions**

* NucleoSpin® Gel and PCR Clean-up by MACHEREY-NAGEL
* [NucleoSpin® Gel and PCR Clean-up](http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)

5. **DNA assembly**

* NEBuilder® HiFi DNA Assembly Cloning Kit
* [NEBuilder® HiFi DNA Assembly Cloning Kit](https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)

6. **Enzymes for DNA analysis and manipulation**

* CopI (RsrII) by Thermo Scientific
* [CopI (RsrII)](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012193_CpoI_RsrII_10_UuL_200U_UG.pdf)
* Ero32I (EcoRV) by Thermo Scientific
* [Ero32I (EcoRV)](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012443_FastDigest_Eco32I_400uL_UG.pdf)
* KspAI (HpaI) by Thermo Scientific
* [KspAI (HpaI)](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012523_FastDigest_KspAI_UG.pdf)
* PstI by Promega
* [PstI](https://www.promega.co.uk/products/cloning-and-dna-markers/restriction-enzymes/psti/?catNum=R6111)
* SpeI by Promega
* [SpeI](https://www.promega.co.uk/products/cloning-and-dna-markers/restriction-enzymes/spei/?catNum=R6591)

#### [Fluorescence Microscopy](#P22-collapse)

**Fluorescence Microscopy**

1. **Scrape off a small portion of a colony from the transformation plate.**
2. **Use a sterile toothpick to streak cells on an object slide.**
3. **Add a small drop of water and cover with a cover slip.**
4. **Put the slide in the holder with the cover slip facing downwards.**

* *Note the switch on the microscope that controls the backlight. This should be turned off to see fluorescence.*

5. **Start at low magnification and increase, focusing on the smaller groups of cells.**
6. **Once a target is selected, put immersion oil on the 100x objective lens and put it in place.**
7. **The image can be diverted from eyepiece to screen if required.**

* *Images can then be captured and saved.*

* ABOUT US
* We are an overgraduate team representing the University of York.
* Our Quicker Way to Analyse Co-Cultures (QWACC) is our entry into the Hardware track for iGEM 2017.

* LOCATION
* Department of Biology
* [University of York](//www.york.ac.uk)
* Heslington
* North Yorkshire
* YO10 5DD

* CONTACT US
* Email: igemyork@gmail.com
* You can also reach us through our social media via the links below.
* [![](//2017.igem.org/wiki/images/0/0c/Icon-facebook.png)](//www.facebook.com/igemyork/)
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# Team:York/Results

[YORK](//2017.igem.org/Team%3AYork)

* [HOME](//2017.igem.org/Team%3AYork)
* [PROJECT](////2017.igem.org/Team%3AYork/Description)

  + [DESCRIPTION](//2017.igem.org/Team%3AYork/Description)
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  + [RESULTS](//2017.igem.org/Team%3AYork/Results)
  + [BACKGROUND](//2017.igem.org/Team%3AYork/Background)
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* [DIHM](https://2017.igem.org/Team%3AYork/Hardware)

  + [HARDWARE](https://2017.igem.org/Team%3AYork/Hardware)+ [SOFTWARE](https://2017.igem.org/Team%3AYork/Software)
    + [MEASUREMENT](https://2017.igem.org/Team%3AYork/Measurement)
* [COMMUNITY](https://2017.igem.org/Team%3AYork/Human_Practices)

  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AYork/Human_Practices)
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  + [HP GOLD INTEGRATED](https://2017.igem.org/Team%3AYork/HP/Gold_Integrated)
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* [OUR LAB](https://2017.igem.org/Team%3AYork/Achievements)

  + [NOTEBOOK](https://2017.igem.org/Team%3AYork/Notebook)
  + [PROTOCOLS](https://2017.igem.org/Team%3AYork/Experiments)
  + [SAFETY](https://2017.igem.org/Team%3AYork/Safety)
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* [ABOUT US](https://2017.igem.org/Team%3AYork/Team)

  + [MEET THE TEAM](https://2017.igem.org/Team%3AYork/Team)
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# Results

# Our Results

Detailed, below, are the results we obtained during the various experimental stages of this project. Accompanying each display of data is a small paragraph of conclusive remarks which aims to outline the importance or significance of the results.
You can get straight to a section by following these links:

* [Modifying *Chlamydomonas reinhardtii*](//2017.igem.org/Team%3AYork/Results#Modifying-Chlamydomonas)
* [Growth Tests](//2017.igem.org/Team%3AYork/Results#Growth-Tests)
* [Constructs](//2017.igem.org/Team%3AYork/Results#Constructs)
* [Size and Shape Analysis](//2017.igem.org/Team%3AYork/Results#Sizes)
* [Cell Density Analysis](//2017.igem.org/Team%3AYork/Results#Cell-Counts)
* [Motility Observations](//2017.igem.org/Team%3AYork/Results#Motility)
* [Conclusions](//2017.igem.org/Team%3AYork/Results#Conclusions)

# Modifying *C. reinhardtii*

We attempted to introduce MEX-1, a maltose exporter gene normally found in the plastid membrane, into the cell membrane of *C. reinhardtii*.

Unfortunately, the results were bleak.

The GC-content of *C. reinhardtii*’s genome is around 60% [1]. This is a likely cause of the modification’s failure, since high GC content is linked with difficulty in amplifying through PCR [2].

Further, despite our best attempts at performing the transformation, we simply did not have enough time to get a successful result. *C. reinhardtii* has a relatively slow growth rate (doubling around once every 8 hours at 25°C [3]) which, coupled with the waiting time for sequencing results, meant that, over the short period in which we could use laboratories in our Department of Biology, we only had time for two attempts at modification. Both were unsuccessful and, thence, we had to abandon the idea that *C. reinhardtii* could export maltose as a carbon source for LW06.

# Will it grow, if...?

Concurrently with the above, we tested the growth of our organisms under different circumstances and on different media to see what ramifications the effects would have for our proposed co-culture.

**LW06 on Maltose**

* + ![](//2017.igem.org/wiki/images/9/93/IGEM-York-LW06-Maltose-Fig1.png)
  + **Figure 1:** Maltose test plate 1 box plot. Plate 1 has serious anomalies in the blanks and controls when compared to plates 2 and 3. RV on y axis is the regression values. Results from this experiment where discounted due to problems with the control and blank samples.

  + ![](//2017.igem.org/wiki/images/7/7c/IGEM-York-LW06-Maltose-Fig2.png)
  + **Figure 2:** Maltose test plate 2 box plot. Missing data points in the blanks and controls are due to OD data not being high enough to be logged by R studio software. RV on y axis is the regression values.
* + ![](//2017.igem.org/wiki/images/2/24/IGEM-York-LW06-Maltose-Fig3.png)
  + **Figure 3:** Maltose test plate 3 box plot. RV on y axis is the regression values.

  + ![](//2017.igem.org/wiki/images/1/10/IGEM-York-LW06-Maltose-Fig4.png)
  + **Figure 4:** Maltose test plate 1 column graph, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells. Given previously described issues with controls data from this experiment is not to be used for analysis.
* + ![](//2017.igem.org/wiki/images/2/26/IGEM-York-LW06-Maltose-Fig5.png)
  + **Figure 5:** Maltose test plate 2 column graphs, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells.

  + ![](//2017.igem.org/wiki/images/7/77/IGEM-York-LW06-Maltose-Fig6.png)
  + **Figure 6:** Maltose plate 3 column graph, mean growth rates on maltose gradient in 96 well plates. Media maltose (1-8) represents columns 5–12 (TP.Maltose) wells.

**Results:** Consistency is shown for high growth in column 5 (1g/L) but remains more or less level throughout the increasing maltose concentrations. Such consistency indicates LW06 is somewhat poor at maltose metabolism but is able to survive on it. Mean growth rates are small, though this is potentially explained by a highly limiting culture volume. However, data from the plate reader indicates clear exponential and plateau events during growth. In conclusion, growth under maltose conditions is possible. Conditioning for maltose consumption may be required to improve final culturing with *C. reinhardtii* when it is exporting maltose, in future co-cultures. The problems with the first experimental run, contamination in the blanks and controls, were most likely due to condensation on the interior lid or displacement of culture material in the controls and blank sections of the plate during agitation. As a result of this, data from this experiment is to be discounted for analysis and should be considered when proceeding with experiments in the future as to avoid issues of contamination.

**LW06 on Home Blend Medium**

* + ![](//2017.igem.org/wiki/images/a/a8/IGEM-York-LW06-Hblend-Fig1.png)
  + **Figure 7:** Home blend (Hblend) test plate 1. Regression values (y axis) show that TP-Maltose performs worse than TP-Glucose. Hblend-Glucose greatly out performs Hblend-Maltose, but is comparable to TP-Maltose.

  + ![](//2017.igem.org/wiki/images/9/9e/IGEM-York-LW06-Hblend-Fig2.png)
  + **Figure 8:** Hblend test plate 1. Mean growth rates here show that the growth rate for Hblend-Glucose is much greater than TP or Hblend-Maltose. TP-Maltose outperforms our Home blend recipe.
* A T-test was conducted for TP.Maltose and Hblend.Maltose. T-test (Degrees of Freedom = 48, p = 8.82 x 10-10). This indicates significant difference between media types.
* + ![](//2017.igem.org/wiki/images/f/fa/IGEM-York-LW06-Hblend-Fig3.png)
  + **Figure 9:** Hblend test plate 2. Regression values (y axis), as in figure 7, show that TP and Hblend-Maltose are out performed by Hblend-Glucose.

  + ![](//2017.igem.org/wiki/images/3/31/IGEM-York-LW06-Hblend-Fig4.png)
  + **Figure 10:** Hblend test plate 2. Mean growth rates here show Hblend-Glucose out performs TP or Hblend-Maltose with no difference between the latter pair in terms of growth.
* A T-test was conducted for the second plate. T-test (D.o.F. = 48, p = 0.058255). For the second plate, no statistical significance between media types was indicated.

**Results:** Overall, Hblend performs unremarkably when compared to TP media. Despite having much higher micronutrients, Hblend does not perform as well as we hypothesised. Some data collected indicates that a longer run time may have had an effect, but it is unlikely that this would lead to major improvements over the TP media. The problem seems to be the uptake or metabolism of maltose. Conditioning our organisms to grow on maltose may improve growth rates in a co-culture. However, such a limitation may work to the advantage of the co-culture. A key problem to consider was the greatly divergent growth rates between *C. reinhardtii* and the LW06 *E. coli* strain. This reduced growth with maltose as the carbon source may provide a sufficient rate limiter on the growth of LW06 to avoid complete culture saturation with the *E. coli* in future co-cultures.

***C. reinhardtii* Ethanol Tolerance**

* + ![](//2017.igem.org/wiki/images/6/6e/IGEM-York-Chlamy-Ethanol-Fig1.png)
  + **Figure 11:** Cell counts for *C. reinhardtii* via multiple well plate readers. Readings taken ~24hours manually.

  + ![](//2017.igem.org/wiki/images/3/30/IGEM-York-Chlamy-Ethanol-Fig2.png)
  + **Figure 12:** Cell counts for *C. reinhardtii* via multiple well plate readers. Readings taken ~24hours manually.

**Results:** *C. reinhardtii* is able to tolerate an increase of ethanol to at the most 3.2% (as a portion of media). Given rates of increase it seems that it could tolerate much higher concentrations. This is encouraging, as ethanol resistance of the *C. reinhardtii* would likely be sufficient to survive upper limits of the LW06 strain’s ethanol production. Interestingly, *C. reinhardtii* experiences better growth at higher concentrations of ethanol, there is not a clear reason this is the case. Addition of ethanol to the media may have made growth conditions more amenable to *C. reinhardtii*. There is some chance that the ethanol may have evaporated which, while possible, is unlikely due to the sealing of the plate. Overall, it seems that *C. reinhardtii* would be able to survive the proposed amount of ethanol that could be produced by the LW06.

# Co-culture of *C. reinhardtii* and *E. coli* LW06

There's a large growth disparity between the two organisms [3][4], so we also performed an experiment to check if LW06 would vastly outperform *C. reinhardtii* on all types of media.

**Growth in a Co-culture**

* + ![](//2017.igem.org/wiki/images/7/76/IGEM-York-Co-culture-Ratio-Experiment-Fig1.png)
  + **Figure 13:** Cell counts for LW06 in each culture via spread plating at 104 dilutions.

  + ![](//2017.igem.org/wiki/images/6/64/IGEM-York-Co-culture-Ratio-Experiment-Fig2.png)
  + **Figure 14:** Cell counts for *C. reinhardtii* via manual haemocytometer microscope cell counting at a 104 dilution.
* + ![](//2017.igem.org/wiki/images/8/86/IGEM-York-Co-culture-Ratio-Experiment-Fig3.png)
  + **Figure 15:** Overlaid cell counts for the co-culture. LW06 represented by blue points and *C. reinhardtii* by orange.

**Results:** Overall LW06 outperforms *C. reinhardtii* in all but one of the cultures (Tube 4). It is encouraging to see LW06 perform well on TP.Maltose and TAP.Maltose, although the latter may have been due to the presence of a second carbon source in the form of acetate. It is odd that in every one of the experiments tube that were inoculated with 100 μl of 0.5-1.0 OD LW06 that the cell counts are considerably lower. It seems that the initial starting cell density may have inhibited growth but it is not clear why this effect would continue when dispersed in the co-culture. Alternatively, an unknown error may have occurred at some point during inoculation. From the results it is clear that, if it was some unknown or unnoticed error, it was made consistently in every one of the co-cultures with 100 μL inoculation. No relevant conclusions can be drawn from this. However, considerations such as the growth limiting factor of the TP.Maltose may work in our interest as to avoid culture saturation with LW06.

An obvious error, in hindsight, is the issue of the 10 μL inoculation. It is likely that a much higher volume added to the culture would have improved the results. This would have provided a high enough cell density, should growth have not be great enough during co-culturing with the modified *C. reinhardtii* in TP media. Overall co-cultures with these two organisms are possible, but higher OD/volume of inoculant may be needed.

# Constructs

We constructed several plasmids to attempt the introduction of MEX-1 into the outer membrane of *C. reinhardtii*. The figures, below, show maps of these plasmids and our evidence that we correctly made the constructs.

### **Plasmid Maps**

#### [**Figure 16:** Backbone pLM005](#Map1-collapse)

#### [**Figure 17:** pLM005 + MEX-1](#Map2-collapse)

#### [**Figure 18:** pLM005 + MEX-1 + Signal Peptide](#Map3-collapse)

**Results:** We used plasmid pLM005 as our vector (see figure 16), which is designed specifically for the expression of eukaryotic ORFs and incorporates a YFP fusion at the C terminal end of the recombinant protein, so we could detect localisation of our edited MEX-1 to the outer membrane.
Cutting of the plasmid at the two HpaI sites removes a small fragment of the plasmid and provides the insertion site for the recombinant gene. Once amplified, the vector is cut again at the eco-RV sites (not shown) to produce a cassette for transformation directly into the *C. reinhardtii* genome.
We successfully cloned and inserted the MEX-1 gene into the pLM005 vector (see figure 17, gel electrophoresis and fluorescent microscopy images below). Our strategy for relocation of MEX-1 (which is a chloroplast membrane protein [5]) was to remove the signal peptide of MEX-1 by PCR cloning and assemble the truncated MEX-1 gene with the signal peptide sequence of known outer membrane protein ACA3 (see figure 18). We were able to successfully assemble this construct (see gel electrophoresis), however we did not manage to successfully transform it into *C. reinhardtii*.

### **BioBrick**

#### [**Figure 19:** BioBrick](#Map5-collapse)

#### [**Figure 20:** BioBrick Fragments](#Map6-collapse)

#### [**Figure 21:** BioBrick + MEX-1 Insert](#Map7-collapse)

**Results:** We designed a biobrick based on the pLM005 plasmid which we used for our project (described above). It uses the silver RFC[23] prefix and suffix for insertion of a eukaryotic ORF into that site where it is tagged at the C terminal end by a YFP and expression is driven by the strong, constitutive PsaD promoter (see figure 19). We used the complimentary 1000bp gblock offer from IDT to synthesise fragments of our biobrick with 18bp overlaps for Gibson assembly. However, this large construct required 7 fragments (see figure 20) and we were unsuccessful in our attempts to assemble.

### **Evidence**

#### [**Figure 22:** Gel Electrophoresis](#Map4-collapse)

* ![](//2017.igem.org/wiki/images/c/c0/IGEM-Gel-1-MEX-1.png)
* **A:** Gel electrophoresis of PCR cloned genes from plasmid library of *C. reinhardtii* genome. ACA3 TMD is the region of the ACA3 gene 5' of the first transmembrane domain, ACA3 SP is the region of the ACA3 gene predicted to code the signal peptide.

* ![](//2017.igem.org/wiki/images/a/a9/IGEM-Gel-2-MEX-1.png)
* **B:** Gel electrophoresis of PstI digested MEX-1 plasmids after Hifi assembly. Colonies 1,3,4,6 &10 appear to contain successfully assembled constructs.

#### [**Figure 23:** Fluorescence Microscopy](#Evidence-collapse)

* ![](//2017.igem.org/wiki/images/3/3f/IGEM-York-WT-no-filter.tif)
* **A:** Wild-type *Chlamydomonas reinhardtii* under a fluorescence microscope before filtering for the Venus fluorescence wavelength.

* ![](//2017.igem.org/wiki/images/3/35/IGEM-York-WT-filter.tif)
* **B:** The wild-type under fluorescence microscope with a filter for the Venus fluorescence wavelength.

* ![](//2017.igem.org/wiki/images/8/80/IGEM-York-VC-no-filter.tif)
* **C:** *C. reinhardtii* after our insertion of the Venus tag, before wavelength filtering.

* ![](//2017.igem.org/wiki/images/3/3c/IGEM-York-VC-filter.tif)
* **D:** *C. reinhardtii* after our insertion of the Venus tag, after wavelength filtering.

**Results:** Figure 22 shows our gel electrophoresis data, confirming that we correctly assembled our construct. Figure 23 shows that we were able to insert our Venus control into *C. reinhardtii*. Unfortunately, the same was not true of our MEX-1 construct.

# Size and Shape Analysis

Though we had some trouble with alignment of lenses, we achieved several results wherein observing the shape of an organism in the focal plane was facilitated. We present the results below. Organisms are outlined in red boxes.

**Confirming Shapes and Sizes of Organisms**

* + ![](//2017.igem.org/wiki/images/4/4c/IGEM-York-BW25113-Shape.png)
  + **Figure 24:** *E. coli* strain BW25113. Its size is (6 x 4) pixels. This corresponds to (1.34 x 0.90) µm.

  + ![](//2017.igem.org/wiki/images/4/4e/IGEM-York-BW25113-Shape2.png)
  + **Figure 25:** *E. coli* strain BW25113. Its size is (9 x 6) pixels. This corresponds to (2.02 x 1.34) µm.
* + ![](//2017.igem.org/wiki/images/a/af/IGEM-York-ChlamyColi-Shape.png)
  + **Figure 26:** This sample contained wildtype *E. coli* and *C. reinhardtii*. It was provided by Dr. Laurence Wilson, captured using his own DIHM. The hologram was produced using our software and the size of *E. coli* was (6 x 4) pixels, which equates to (1.34 x 0.90) µm.

  + ![](//2017.igem.org/wiki/images/5/50/IGEM-York-Chlamydomonas-Shape.png)
  + **Figure 27:** *Chlamydomonas reinhardtii*. The sizes were as follows. A: (9 x 10) pixels, (10.1 x 11.2) µm. B: (9 x 9) pixels, (10.1 x 10.1) µm. C: (10 x 9) pixels, (11.2 x 10.1) µm. D: (10 x 9) pixels, (11.2 x 10.1) µm.

**Results:** These results show that our software is capable of producing holograms within which the shapes of organisms can be distinguished. In figures 24 to 26, the *E. coli* can be seen to be rod shaped, approximately 2 µm in one direction and 1 µm in the other, as expected [6]. In the figure 27, *C. reinhardtii* is approximately circular with diameter around 10 µm, also as expected [3]. These are promising results which show the potential for the technique to be used to distinguish between organisms within a co-culture, especially when there is a size discrepancy. This is exemplified in figure 26, most of all. Figures 24, 25 and 27 are also evidence that the quality of images taken with our hardware is sufficient for shape detection. As seen [here](//2017.igem.org/Team%3AYork/HP/Silver#International), we also tested the ability to find the size and shape of organisms with *Saccharomyces cerevisiae*, which is of an intermediate size when compared with *E. coli* and *C. reinhardtii*. The *S. cerevisiae* was kindly provided by the iGEM Aachen 2017 team.

# Cell Density Analysis

We used our analysis software to count the cells in manually-selected focal planes. All of the below images are from data taken with our own hardware and software. We then calculated the volume of the hologram in each case (accounting for magnification, step size and number of steps) and used both to find the number of cells per millilitre. We used the formula: Cells/mL = (D x C x 10-6) / (5122 x N x ((1.12/M)x10-6)3 ); wherein C is the counted number of blobs from the analysis software, D is the dilution number compared with the corroborated sample (e.g. D = 100 for a 1 part in 100 dilution, D = 1 for an undiluted sample), M is the magnification and N is the number of steps between the focal plane being analysed and the closest other plane with focused cells. In most cases N was 150, indicating the whole hologram had only one focused section. We corroborated our values' relevance by comparing to either an optical density reading or a reading from a ThermoFisher Countess II FL Automated Cell Counter.

**Confirming Cell Counts**

* + ![](//2017.igem.org/wiki/images/7/77/IGEM-Chlamy-6e5-count-13.png)
  + **Figure 28:** *C. reinhardtii*: C = 13, D = 10, M = 1, N = 150. The Countess II FL gave a reading of (5.19 x 106) cells/mL. Our calculated value was (2.4 x 106) cells/mL.

  + ![](//2017.igem.org/wiki/images/4/4b/IGEM-Chlamy-no-dilution-5x-count-13.png)
  + **Figure 29:** *C. reinhardtii*: C = 113, D = 1, M = 5, N = 150. The Countess II FL gave a reading of (2.03 x 108) cells/mL. Our calculated value was (2.6 x 108) cells/mL.

  + ![](//2017.igem.org/wiki/images/9/9a/IGEM-TAP-count-10.png)
  + **Figure 30:** TAP medium only: C = 10, D = 1, M = 1, N = 150. There are no microorganisms present, the diffraction patterns here are due to dust on the slide. The Countess II FL gave a reading of (1.47 x 104) cells/mL. Our calculated value was (1.8 x 105) cells/mL.
* + ![](//2017.igem.org/wiki/images/e/e7/IGEM-E-Coli-1in100-count-10.png)
  + **Figure 31:** *E. coli*: C = 10, D = 100, M = 5, N = 150. The foci were slightly misshapen (but still countable) due to misalignment of the lens during setup. The OD reading returned (4 x 109) cells/mL. Our calculated value was (2.3 x 109) cells/mL.

  + ![](//2017.igem.org/wiki/images/d/dc/IGEM-Gal-7e6-count-248.png)
  + **Figure 32:** *Galdieria sulphuraria*: C = 248, D = 1, M = 1, N = 150. The Countess II FL reading returned (5.3 x 106) cells/mL. Our calculated value was (4.5 x 106) cells/mL.

**Results:** **Figure 28** shows a well focused, not-magnified image of *C. reinhardtii*. It is clear from the image that several cells that were present within the area of the image were not detected. However, many, if not all, of them did not pass through the focus of the constructed hologram. Further, the cell count we obtained was only a factor of 2 different to that given by an lab-grade automated cell counter. Considering the minimally developed analysis software, this is a promising achievement that could likely be improved with time and better algorithms.

**Figure 29** shows a 5x magnified image of *C. reinhardtii*. The diffraction patterns are misshapen (we expect circles, like in figure 28, but larger). This is due to the lens being slightly misaligned, which points to a weakness of the hardware's design. Perfect alignment is difficult to achieve when the levelness of component platforms must be adjusted by hand. It can be seen, from the figure, that some of the detected blobs are in erroneous positions and that some cells remain undetected. Upon counting manually, it seems that these two errors roughly cancel each other out. We conclude from this figure that the design of the hardware and software must be improved before industrial use is realisable, though this (and our other data) serve as proof of concept.

**Figure 30** shows a not-magnified image of a slide whose contents is solely TAP medium. The diffraction patterns that are apparent are most likely caused by dust or imperfections in the slide. The count we obtained for this hologram was an order of magnitude higher than the equivalent result from the automated cell counter, but was an order of magnitude (or more) lower than that for other not-magnified holograms that we took, even the least populous. The conditions used for detection in this image were the same as for figure 28, showing that our uncertainty in a measurement like this is likely around 10%. This is twice that of the automated cell counter we used [7]. This is a positive result, as it implies that, as the analysis techniques we have used are developed in future, we can approach the accuracy of widely used lab equipment.

**Figure 31** shows *E. coli* LW06 at 5x magnification. The cells' diffraction patterns were misshapen for the same reasons as those presented, above, for figure 29. Regardless, the count achieved was around a factor of 2 less than the value obtained through optical density analysis (which was performed at 600 nm wavelength). Yet again, as evidence that our concept is useful, this result is promising.

**Figure 32** shows *Galdieria sulphuraria* at 1x magnification. This was a densely populated sample, though the software still performed well. The blue outline denotes that the cell was detected by looking at the gradient image. The percentage difference between the Countess II FL reading and our own value was 15%. This equates to a difference in count of around 7. This serves as perhaps our best evidence that, when all goes according to plan with our current hardware and software, it functions as an at least somewhat viable alternative to other cell counting methods.

**Confirming Cell Counts in a Co-Sample**

* + ![](//2017.igem.org/wiki/images/5/59/IGEM-GaldieriaChlamy-whole-range-count-21.png)
  + **Figure 33:** *C. reinhardtii* and *G. sulphuraria*: C = 21, D = 1, M = 1, N = 150. The Countess II FL reading returned (6.28 x 105) cells/mL for the combined total of the organisms. Our calculated value was (3.8 x 105) cells/mL.

  + ![](//2017.igem.org/wiki/images/f/fd/IGEM-GaldieriaChlamy-high-range-count-9.png)
  + **Figure 34:** *C. reinhardtii* and *G. sulphuraria*: C = 9, D = 1, M = 1, N = 150. The Countess II FL was only capable of reading the total cell count, so we have no corroboratory datum for this test. Our count for the larger species was (1.6 x 105) cells/mL.

**Results:** Figures 33 and 34 show a co-sample of *C. reinhardtii* and *G. sulphuraria*. It was made by combining a sample of a set volume for each of the organisms. The *C. reinhardtii* sample had (4.17 x 105) cells/mL and the *G. sulphuraria* had (3.02 x 105) cells/mL. We suppose, therefore, that *Galdieria* made up 42.0% of the resultant co-sample's organisms by number. Our count from figure 34 is 42.1% of the count in figure 33. Figure 33's count was found by considering the entire range of documented radii for both organisms [7][8]. Figure 34's count was the upper half of that range, within which *G. sulphuraria* has been documented to reach during its lifecycle [8]. We conclude that it is most likely that we have found the ratio between two organisms of slightly different size in a co-sample. This, we present as evidence that our microscope and software are a useful tool for the determination of cell density in samples containing multiple visibly distinguishable organisms.

In short, the above data evidences the promising potential of our software and hardware combination with respect to use as a non-intrusive and fast method of monitoring the growth of cells in monocultures and in co-cultures. All of the image processing and calculations as outlined above can be done, in minutes, on the same Raspberry Pi that is used to take the images.

# Motility Observations

Below we present evidence that our microscope is capable of showing the movement of organisms. Unfortunately, *C. reinhardtii* was the only organism we captured moving autonomously. We also tried to modify *E. coli* to increase its motility (see the [Description](//2017.igem.org/Team%3AYork/Description) page), though this was unsuccessful. We borrowed another strain (DB5) that moves a lot, too. When we tried to look at it using our microscope, we couldn't see any moving cells, despite being able to see them under a light microscope. We did capture footage of *E. coli* moving due to the flow of liquid medium, however. It is presented below, also.

*Chlamydomonas reinhardtii* Moving in Samples

* ![](//2017.igem.org/wiki/images/3/39/IGEM-Chlamy-Movement-GIF.gif)
* Figure 35: A GIF of a *C. reinhardtii* cell moving in a sample at 5x magnification.

* [](//2017.igem.org/wiki/images/f/f7/IGEM-Chlamy-Movement-VID.mp4)
* Figure 36: A video showing a *C. reinhardtii* cell moving in a sample at 5x magnification.

**Results:** It is apparent that the cells, above, are moving autonomously when compared with the motion due to flow as seen for *E. coli* below. The movement of *C. reinhardtii* is considerably more erratic and does not follow a single line/smooth path.

*E. coli* DB5 Drifting with Flow of Medium

* [](//2017.igem.org/wiki/images/5/5f/IGEM-DB5-Movement-VID.mp4)
* Figure 37: A video showing *E. coli* cells moving, from the top-right in a down/left direction, in a sample at 5x magnification.

**Results:** Though the moving diffraction patterns are faint, it cn be seen that the movement of each cell in the above video is identical, smooth and in a single direction. Hence, we assert that the motion is drift, rather than autonomous transit.

# Conclusions

In summary, we can draw several conclusions. We have had some successes and have seen some areas that seem promising but require more work.

The main shortcomings:

* Creating a closed analysis system using a milli-fluidic chamber.
* *We came to realise that we required a thinner analysis chamber. Some evidence can be found [here](//2017.igem.org/Team%3AYork/Design#Milli-fluidic-Problem).*
* Inserting MEX-1 into the outer membrane of *Chlamydomonas reinhardtii*.
* *Our transformation of* C. reinhardtii *failed. As mentioned [here](//2017.igem.org/Team%3AYork/Results#Modifying-Chlamydomonas).*
* Quantifying the motility of organisms using our DIHM setup.
* *We could see* C. reinhardtii *moving on various occasions, but failed to observe the motility of* E. coli *DB5, a motile strain. As discussed [here](//2017.igem.org/Team%3AYork/Results#Motility).*
* Using a UI to control the DIHM from a third party computer.
* *We had a great design, there was a compatibility bug that we could not fix in the time we had. As seen [here](//2017.igem.org/Team%3AYork/Design#UI-Problem).*

The main successes:

* We created inexpensive and functioning upright DIHM hardware and open source holography software.
* *The hardware can be seen [here](//2017.igem.org/Team%3AYork/Hardware). The software is discussed [here](//2017.igem.org/Team%3AYork/Software).*
* We created blob detection software which is capable of finding cells of a given size in a focused image.
* *The software is mentioned in more detail [here](//2017.igem.org/Team%3AYork/Software#Blob-Detection).*
* We successfully observed the shapes of organisms using our DIHM and holography software.
* *This can be seen [here](//2017.igem.org/Team%3AYork/Results#Sizes).*
* We calculated the cell density of samples consistently to a fair degree of accuracy, using our DIHM and holography software.
* *As shown [here](//2017.igem.org/Team%3AYork/Results#Cell-Counts).*
* We confirmed that *C. reinhardtii* and *E. coli* can grow together in a co-culture.
* *This is evidenced [here](//2017.igem.org/Team%3AYork/Results#Growth-Tests).*
* We successfully constructed our MEX-1 plasmid.
* *Though we didn't manage to make* C. reinhardtii *accept it, we present proof that we correctly constructed it [here](//2017.igem.org/Team%3AYork/Results#Constructs).*

Hence, our overarching conclusion is that digital holography is not only low-cost, easily adapted and useful for analysing co-cultures but also holds great potential in terms of future applications. We mention one such future application on our [Applied Design](//2017.igem.org/Team%3AYork/Applied_Design#Future-Application) page.

# References

* [1]: S. Merchant, et al. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions, Science
* [2]: Y. Benita, et al. Regionalized GC content of template DNA as a predictor of PCR success, Nucleic Acids Research
* [3]: E. H. Harris, The Chlamydomonas Sourcebook, 2nd Edition
* [4]: A. G. Marr, Growth rate of Escherichia coli, Microbiological Reviews
* [5]: T. Niittylä, A Previously Unknown Maltose Transporter Essential for Starch Degradation in Leaves, Science
* [6]: J. H. Cummings and G. T. Macfarlane, Role of intestinal bacteria in nutrient metabolism, Journal of parenteral and enteral nutrition
* [7]: Countess II FL Automated Cell Counter, ThermoFisher Scientific, as available on 31st October 2017 at [www.thermofisher.com/order/catalog/product/AMQAF1000](//www.thermofisher.com/order/catalog/product/AMQAF1000)
* [8]: P. Albertano, C. Ciniglia, G. Pinto & A. Pollio, The taxonomic position of Cyanidium, Cyanidioschyzon and Galdieria: an update, Hydrobiologia

* ABOUT US
* We are an overgraduate team representing the University of York.
* Our Quicker Way to Analyse Co-Cultures (QWACC) is our entry into the Hardware track for iGEM 2017.

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