iGEM 2019 — Team Edinburgh_UG

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# Team:Edinburgh UG/Notebook

* [Team Members](https://2019.igem.org/Team%3AEdinburgh_UG/Team)
* [Collaborations](https://2019.igem.org/Team%3AEdinburgh_UG/Collaborations)

* [Description](https://2019.igem.org/Team%3AEdinburgh_UG/Description)
* [Design](https://2019.igem.org/Team%3AEdinburgh_UG/Design)
* [Experiments](https://2019.igem.org/Team%3AEdinburgh_UG/Experiments)
* [Model](https://2019.igem.org/Team%3AEdinburgh_UG/Model)
* [Notebook](https://2019.igem.org/Team%3AEdinburgh_UG/Notebook)
* [Results](https://2019.igem.org/Team%3AEdinburgh_UG/Results)
* [Attributions](https://2019.igem.org/Team%3AEdinburgh_UG/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AEdinburgh_UG/Parts)
* [Basic Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Basic_Part)
* [Composite Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AEdinburgh_UG/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AEdinburgh_UG/Public_Engagement)

* [Medals](https://2019.igem.org/Team%3AEdinburgh_UG/Medals)
* [Awards](https://2019.igem.org/Team%3AEdinburgh_UG/Awards)

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# Notebook

### Week 1 03/06/19 - 07/06/19

This week was spent brainstorming ideas for our project this summer. Everyone in the team was involved with research and development of different topics suggested. At the end of the week, we had split into two groups: one researching bio-calculators and the other focused on biohydrogen.

### Week 2 10/06/19 - 14/06/19

Biohydrogen is voted as the project and the whole team comes back together. Further research is undertaken by everyone, with special focus on previous iGEM projects. Our design process begins.

### Week 3 17/06/19 - 21/06/19

Our research led us to iGEM team Macquarie University 2017 who worked with *Chlamydomonas reinhardtii* hydrogenase (Hyd1), as well as the *Pyrococcus furiosus* hydrogenase (Sh1) from literature. We decide on *Escherichia coli* and *Rhodobacter sphaeroides* as our chassis. We start designing our gene constructs, vectors and transformation methods.

### Week 4 24/06/19 - 28/06/19

This week was spent finalising our genetic constructs as well as researching more into how to culture *R. sphaeroides*. This is our first week doing wet lab work; inoculation of LB with *E. coli* DH5-alpha and incubated overnight at 37℃.

### Week 5 01/07/19 - 05/07/19

CIDAR MoClo kit was received as *E. coli* cells, and this was incubated at 37℃. *R. sphaeroides* 2.4.1 wild-type strain was also received.

03/07: Cells were cultured for plasmid purification → 5ml LB (100 µg/ml Amp or 50 µl/ml Kan). *R. sphaeroides* strain was cultured on LB plates (1 µg/ml tetracycline) overnight at room temperature. Little growth was seen the following morning.

04/07 - 05/07: CIDAR plasmids were purified, digested and ran on a gel. There were 10 plasmids in total, each containing a different part: A1, A7, B1, B7, D7, E5, H4, H6, H7, and H8. Preparation of CaCl2 and MgCl2 buffers for chemically competent cells. *R. sphaeroides* was again plated and incubated at 30℃ over the weekend.

The design of our genetic constructs were finalised:

Hyd1 & Ferredoxin clusters

![](https://static.igem.org/mediawiki/2019/f/f3/T--Edinburgh_UG--note1.png)

![](https://static.igem.org/mediawiki/2019/b/b1/T--Edinburgh_UG--note2.png)

Sh1 & Associated Maturation Proteins clusters

![](https://static.igem.org/mediawiki/2019/3/31/T--Edinburgh_UG--note3.png)

![](https://static.igem.org/mediawiki/2019/6/6f/T--Edinburgh_UG--note4.png)

### Week 6 08/07/19 - 12/07/19

Good growth observed on weekend cultures of *R. sphaeroides*. Sistrom’s minimal medium for *R. sphaeroides* was prepared at 10X concentration and stored at 4℃. Ligation of promoter J95022, terminator B1007, E5 (GFP gene), and H7 (plasmid vector) for part characterisation of promoter was done. *E. coli* DH5-alpha competent cells were made, transformed with the ligation mixture, and were plated on X-gal+IPTG+kanamycin plates. Gel electrophoresis of the J95022 promoter+RBS PCR product was carried out. *E. coli* S17-1 competent cells were prepared and stored at -80℃.

### Week 7 15/07/19 - 19/07/19

Gel extraction of J95022+RBS was performed. Level 1 assembly of our genetic parts was carried out, except for the J95025 promoter+RBS+Hyd1 construct. Our initial hydrogen collection method was set up using a water bath at 30℃ and water displacement measurements; the set-up was left for the week to determine its efficacy. Optical density measurements were taken at A660 from R. sphaeroides cultures in LB and Sistrom’s medium to track and compare growth rates. 13 of the transformations using the level 1 ligations were unsuccessful and so needed repeating.

### Week 8 22/07/19 - 26/07/19

PCR amplification of our two level 2 plasmid vectors was carried out (POGG011 & POGG026): 011 was successful but gave weak bands after electrophoresis, and 026 needed repeating. PCR touchdown was done for both plasmids starting at 72℃. Minipreps were prepared from the previous transformations carried out in week 7. The concentrations were measured using a nanodrop machine and the DNA was digested with an endonuclease. The results were visualised on a gel after electrophoresis.

25/07 - 26/07: Team member Jay went to visit Dr Andrew Hitchcock from the University of Sheffield to learn how to transform *R. sphaeroides* efficiently through conjugation with *E. coli*. He also was taught how best to culture the cells and was shown how to prepare M22 growth medium. Jay brought M22 medium back to Edinburgh and this is the medium we now use for *R. sphaeroides*, alongside LB.

### Week 9 29/07/19 - 02/08/19

The gel last week revealed that only 5 out of 14 ligations were successful and so 9 needed repeating. PCR for plasmid 026 was also carried out again, this time with different primer combinations and annealing temperatures. Competent DH5 alpha *E. coli* cells were made (80 at 100 µl) and stored at -80℃.

At the beginning of the week, 6 team members attended the iGEM UK meet-up held at Newcastle University.

### Week 10 05/08/19 - 09/08/19

Ligations and transformations were repeated for those that failed from the previous week. All bar one (number 8) were successful, meaning 13/14 constructs were ready for level 2 assembly.

08/08: We received spent grain, which is a brewery by-product, to test it as a carbon source for our bacteria. We freeze-dried approximately 200 grams of it, ground with a mortar and pestle, steeped in 350 ml of hot water for 2 hours, and filtered to dispose of the hard shells. The pH was brought up from 4.2 to 7.1 using 1M NaOH and trace element solution from YCC medium was added.

### Week 11 12/08/19 - 16/08/19

Ligations for construct 8 were repeated multiple times during the week due to recurring negative results. Alginate gel was prepared for our Hydrolyte artificial leaf design. Polyester fabric was incubated for 1h in 1% PHA 1 mM NaCl solution, washed with 1 mM NaCl and dried. 1 gram of *R. sphaeroides* was mixed with 100 ml 5% sodium alginate solution. The PET fabric was coated and enveloped with the alginate, which was subsequently sprayed with 2% CaCl solution for polymerisation. We prepared 2 gels, one with poly-linen and the other with poly-cotton to compare the fabrics’ abilities to immobilize the cells. A thin layer of M22 medium was poured on top and these were left overnight at 30℃.

### Week 12 19/08/19 - 23/08/19

Alginate gels ‘melted’ and we ran experiments in different conditions to troubleshoot.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Condition | M22; 30℃ | M22; Room Temperature | ΔM22; 30℃ | ΔM22; Room temperature |
| **Outcome** | Melted | Slightly melted | Slightly melted | Solid |

New ligations and minipreps were done on all 14 constructs to ensure that they were all correct. All were successful except for construct number 14 - this was repeated.

PCR was done to remove kanamycin resistance from our constructs as plasmid 026 already carried this gene. Primers with 4 base overhangs were ordered to be added to plasmid 011 as the restriction sites were too close together, hindering the enzyme’s ability to cleave the regions. LacZ was amplified with PCR, phosphorylated with PNK and blunt-end ligated into 026 and 011. This was done because the PCR process to introduce overhangs for efficient digestion kept failing.

### Week 13 26/08/19 - 30/08/19

Restrictions were carried out on 3 samples from plasmids 011 and 026 each.

### Week 14 02/09/19-06/09/09

FDH\_h gBlock arrived from IDT. The transcriptional unit was assembled, transformed into *E. coli* BL21DE3. The correct assembly was confirmed by digesting it SacII and sequencing.
J95022 promoter characterisation was attempted. Cells were grown to 0.6OD, diluted 100 times and induced with 0, 20, 30, 45, 60, 90, 120uM IPTG, fluorescence was measured at 485/520 nm excitation/emission. Only uninduced cells grew.

### Week 15 9/09/19-13/09/09

BL21DE3 cells with FDH were grown overnight, diluted to 0.05 OD600, grown for 2h and then induced with 0.1mM IPTG. Cells were harvested after 4h. SDS-PAGE was run on soluble and insoluble protein fractions to check for protein expression, FDH was not seen on a gel. J95022 promoter characterisation was reattempted. Cells were grown to 0.6OD, and induced with 0, 20, 30, 45, 60, 90, 120uM IPTG, fluorescence was measured at 485/520 nm excitation/emission.

### Week 16. 16/09/2019-20/09/2019

NADH oxidation assay (0.1M pH6.8 Sodium Phosphate buffer, 0.1M bicarbonate, 0.02mM NADH, 0.5mg/ml protein) was tested on soluble and insoluble fractions of BL21DE3 with FDH and compared to BL21DE3 without FDH. Oxidation rate was much higher in FDH soluble fraction. FDH was purified using Ni-resin, purification failed. 026 and 011 plasmids were PCR amplified to introduce BbsI overhangs, treated with DpnI and PNK and ligated. BbsI overhangs were finally successfully introduced.

### Week 17. 23/09/2019-27/09/2019

We repeated chromatography purification and tested protein fractions on SDS-PAGE gel; band of 80.4 kDa was seen. Activity assay was repeated using purified fraction. *R. sphaeroides* constructs ligations were repeated.

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# Team:Edinburgh UG/Experiments

* [Team Members](https://2019.igem.org/Team%3AEdinburgh_UG/Team)
* [Collaborations](https://2019.igem.org/Team%3AEdinburgh_UG/Collaborations)

* [Description](https://2019.igem.org/Team%3AEdinburgh_UG/Description)
* [Design](https://2019.igem.org/Team%3AEdinburgh_UG/Design)
* [Experiments](https://2019.igem.org/Team%3AEdinburgh_UG/Experiments)
* [Model](https://2019.igem.org/Team%3AEdinburgh_UG/Model)
* [Notebook](https://2019.igem.org/Team%3AEdinburgh_UG/Notebook)
* [Results](https://2019.igem.org/Team%3AEdinburgh_UG/Results)
* [Attributions](https://2019.igem.org/Team%3AEdinburgh_UG/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AEdinburgh_UG/Parts)
* [Basic Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Basic_Part)
* [Composite Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AEdinburgh_UG/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AEdinburgh_UG/Public_Engagement)

* [Medals](https://2019.igem.org/Team%3AEdinburgh_UG/Medals)
* [Awards](https://2019.igem.org/Team%3AEdinburgh_UG/Awards)

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* [HOME](https://2019.igem.org/Team%3AEdinburgh_UG)
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* [SAFETY](https://2019.igem.org/Team%3AEdinburgh_UG/Safety)
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* [AWARDS*arrow\_drop\_down*](#!)

# Experiments

### Formate dehydrogenase (FDH)

For our new biobrick contribution, we decided to assemble and characterise formate dehydrogenase (FDH).
This enzyme facilitates this reversible reaction between carbon dioxide and formate: CO2 + NADH ←→ HCOO- + NAD+. Formic acid is a chemical commodity and can be easily stored and transported, making it a stable form of hydrogen fuel.
It can be converted into hydrogen gas by *E.coli*'s native formate hydrogen lyase [footnote 1].
This formate dehydrogenase has high binding affinities for NADH and CO2, and is expected to generate formate efficiently [footnote 2].

The transcriptional unit was assembled using loop assembly. Successful assembly was confirmed using diagnostic digestion and sequencing. His-tagged FDH was over-expressed in *E.coli* strain BL21DE3 using a T7 promoter. Successful expression after induction with IPTG was confirmed by Ni-affinity chromatography column and subsequent SDS-PAGE analysis.

![](https://static.igem.org/mediawiki/2019/4/48/T--Edinburgh_UG--exp1.png)

Activity was assayed through NADH oxidation at 37° and was carried out using purified FDH protein. 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. This showed that recombinant FDH can be expressed in *E.coli* while still maintaining functionality.

![](https://static.igem.org/mediawiki/2019/6/63/T--Edinburgh_UG--exp2.png)

### Brewing co-product - spent grain

![](https://static.igem.org/mediawiki/2019/f/f6/T--Edinburgh_UG--exp6r.png)

We also had the pleasure to receive distillery by-product.
While this made the lab smell a bit like a brewery (and our supervisor wasn't too pleased either...) we were able to prepare the co-product to culture both strains of *Rhodobacter* with the intention of also testing this carbon source with our modified organism.
As reducing costs of our technology is a main aim of our project, we wanted to extend this theme to all aspects of our project’s design.
When speaking with Scottish Gas Network, the ability to use co-product sources in remote areas was flagged as a potential cost cutting solution in certain rural areas.
Find out more about our integrated [Human Practices](https://2019.igem.org/Team%3AEdinburgh_UG/Human_Practices)

### Hydrogen Collection

Our initial setup relied on capturing all gas produced by simple water displacement. This provided a proof of concept and a rough benchmark for how much gas we could expect to produce, but since our culture produced both carbon dioxide and hydrogen this couldn’t give us a quantitative measurement of hydrogen.

Throughout our project we've tried to maintain a theme of frugality as we want to be able to produce a cost effective source of green energy. We thought using a high cost analytical technique such as gas chromatography would go against this; especially since in order to do this it would be necessary to use headspace analysis which would be even more costly. We wanted a cost effective analytical method so we headed out of the lab and back to the drawing board to improve our method.

### The New & Improved

Combining a 2007 Paper by C Zanchetta and colleagues as well as work from the 2017 Macquarie iGEM team allowed for an efficient and cost effective method that produced a quantitative measurement while aligning with our theme of frugality and our human practices [1,2]. This allowed us to use barium hydroxide as Macquarie did to remove carbon dioxide while also being able to carry out hydrogen collection in a closed system.

![](https://static.igem.org/mediawiki/2019/c/c2/T--Edinburgh_UG--exp5.png)

![](https://static.igem.org/mediawiki/2019/d/d8/T--Edinburgh_UG--exp3.png)

When dissolved in water carbon dioxide creates carbonic acid. Carbonic acid reacts rapidly with barium hydroxide forming an extremely insoluble white precipitate; barium carbonate. By passing the gas produced into a flask containing barium hydroxide (0.1 M) it was possible to precipitate carbon dioxide at the interface as barium carbonate.

![](https://static.igem.org/mediawiki/2019/5/51/T--Edinburgh_UG--exp4.png)
Equation 1: Reaction of barium hydroxide and carbon dioxide1

Since the carbon dioxide had been stripped from the gaseous phase the only other gas produced would be hydrogen. The production of hydrogen would lead to an increase in pressure inside the flasks which would, in turn, lead to a force being exerted on the liquid forcing it through the tubing. This results in the water level in the syringe rising in line with the Bernoulli principle. Since under standard conditions an ideal gas takes up 22.4 L this change in water level could then be converted back into moles of hydrogen using the ideal gas law.

![](https://static.igem.org/mediawiki/2019/4/44/T--Edinburgh_UG--exp7.png)

Kieran preparing solution in a fume hood for our new collection methods.

![](https://static.igem.org/mediawiki/2019/b/bd/T--Edinburgh_UG--exp8.png)
Equation 1: Ideal gas law rearranged to give number of moles hydrogen as a function of pressure volume and temperature [1].

However, this equation comes from a system running a catalysed reaction resulting in a far greater change in pressure than will be seen on our scale. As such, it is possible to state that P0 ≈ P(t), this greatly simplifies the equation.

![](https://static.igem.org/mediawiki/2019/d/d7/T--Edinburgh_UG--exp9.png)
Equation 2: Simplified ideal gas law equation for number of moles hydrogen.

The pressure of the system will be equal to atmospheric pressure minus that of the vapour pressure of water at 30 degrees C. This method thereby allowed for cost efficient analysis while also being perfect for the sensitive small scale experiment necessary.

In order to monitor gas production as closely as possible AutoHotKey programming was used to create a script that would take pictures of the syringe at set time intervals [3].

### Promoter Characterisation

We decided to characterise PA1/04/03 lac based synthetic promoter by assembling
it with strong *R. sphaeroides* RBS ([BBa\_J95018](http://parts.igem.org/Part%3ABBa_J95018)),
GFP ([BBa\_E0040](http://parts.igem.org/Part%3ABBa_E0040)) and terminator
([BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006)) using loop assembly. The promoter and RBS were shown to work in *R. sphaeroides*
with high expression rates (Huo, 2011). We tested if this promoter-RBS combination
could yield high expression rates in *E. coli* as this would provide a convenient system
for parallel expression in both *R. sphaeroides* and *E. coli*.
Three 5ml liquid cultures containing recombinant *E.coli* DH5α having
*R. sphaeroides* RBS ([BBa\_J95018](http://parts.igem.org/Part%3ABBa_J95018)), GFP ([BBa\_E0040](http://parts.igem.org/Part%3ABBa_E0040)), terminator ([BBa\_B1006](http://parts.igem.org/Part%3ABBa_B1006)) and
Kanamycin resistance gene were incubated in a 37°C shaker overnight. Next day, these
cultures were diluted to 0.05 OD600 and incubated at 37° to the limit where they had
0.5 OD600. Total of 100μl culture was induced with 8 different IPTG concentrations-
(5μM, 10μM, 20μM, 30μM, 45μM, 60μM, 120μM and 180μM) and measured at an excitation
and emission of 485nm and 520nm. Orbital averaging was set to 3 and the gain to 1300.
Cell growth was measured at 600nm. The fluorescence was divided by the approximated number
of cells to get normalised fluorescence per cell.

![](https://static.igem.org/mediawiki/2019/a/aa/T--Edinburgh_UG--exp10.png)
Figure 1. GFP fluorescence per cell

**References**

1. C Zanchetta, B Patton, G Guella, and A Miotello, 2007, An integrated apparatus for prodyction and measurement of molecular hydrogen, IOP Publishing
2. Macquarie iGEM Team, 2017, HydroGEM
3. https://github.com/A-Sobieska/Edinburgh\_UG\_iGEM\_2019
4. Huo, Junling, "Design of a BioBrickTM Compatible Gene Expression System for Rhodobacter sphaeroides" (2011). All Graduate Theses and Dissertations. 877.
   https://digitalcommons.usu.edu/etd/877

**Footnotes**

1. Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. Enhanced Hydrogen Production from Formic Acid by Formate Hydrogen Lyase-Overexpressing Escherichia coli Strains. Applied and Environmental Microbiology. 2005;71(11):6762-6768. doi:10.1128/aem.71.11.6762-6768.2005
2. Alissandratos A, Kim H-K, Matthews H, Hennessy JE, Philbrook A, Easton CJ. Clostridium carboxidivorans Strain P7T Recombinant Formate Dehydrogenase Catalyzes Reduction of CO2to Formate. Applied and Environmental Microbiology. 2013;79(2):741-744. doi:10.1128/aem.02886-12

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# Team:Edinburgh UG/Results

* [Team Members](https://2019.igem.org/Team%3AEdinburgh_UG/Team)
* [Collaborations](https://2019.igem.org/Team%3AEdinburgh_UG/Collaborations)

* [Description](https://2019.igem.org/Team%3AEdinburgh_UG/Description)
* [Design](https://2019.igem.org/Team%3AEdinburgh_UG/Design)
* [Experiments](https://2019.igem.org/Team%3AEdinburgh_UG/Experiments)
* [Model](https://2019.igem.org/Team%3AEdinburgh_UG/Model)
* [Notebook](https://2019.igem.org/Team%3AEdinburgh_UG/Notebook)
* [Results](https://2019.igem.org/Team%3AEdinburgh_UG/Results)
* [Attributions](https://2019.igem.org/Team%3AEdinburgh_UG/Attributions)

* [Parts Overview](https://2019.igem.org/Team%3AEdinburgh_UG/Parts)
* [Basic Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Basic_Part)
* [Composite Parts](https://2019.igem.org/Team%3AEdinburgh_UG/Composite_Part)
* [Part Collection](https://2019.igem.org/Team%3AEdinburgh_UG/Part_Collection)

* [Human Practices](https://2019.igem.org/Team%3AEdinburgh_UG/Human_Practices)
* [Education & Engagement](https://2019.igem.org/Team%3AEdinburgh_UG/Public_Engagement)

* [Medals](https://2019.igem.org/Team%3AEdinburgh_UG/Medals)
* [Awards](https://2019.igem.org/Team%3AEdinburgh_UG/Awards)

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* [HOME](https://2019.igem.org/Team%3AEdinburgh_UG)
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* [PROJECT*arrow\_drop\_down*](#!)
* [PARTS*arrow\_drop\_down*](#!)
* [SAFETY](https://2019.igem.org/Team%3AEdinburgh_UG/Safety)
* [HUMAN INTEGRATED PRACTICE*arrow\_drop\_down*](#!)
* [AWARDS*arrow\_drop\_down*](#!)

# Results

### Hydrogen Collection Results

![](https://static.igem.org/mediawiki/2019/6/6b/T--Edinburgh_UG--results1.png)

### Formate dehydrogenase (FDH)

We assembled and characterised formate dehydrogenase from Clostridium carboxidivorans. Loop assembly was used to construct the transcriptional unit and transformed into E. coli strain BL21DE3. Successful assembly and transformation was checked through diagnostic digestion and sequencing (figure 1).

![](https://static.igem.org/mediawiki/2019/6/63/T--Edinburgh_UG--results2.png)

![](https://static.igem.org/mediawiki/2019/4/48/T--Edinburgh_UG--exp1.png)
Figure 2: Supernatant (S/N), flow through 1 and 2 (FT1/FT2), and eluted fractions 1 and 2 were run on a 10% SDS-PAGE gel. The expected size of our protein is 80.7kDa.

His-tagged FDH protein expression in E. coli was confirmed through immobilised metal affinity chromatography (IMAC) and subsequent SDS-PAGE analysis. Bands were visualised after destaining and were in the expected region of 80.7 kDa. The concentration of purified protein was determined using Bradford assay and was shown to be 1.8 µM.

Activity was assayed via NADH oxidation at 37° and measuring absorbance at 340nm. This showed that recombinant FDH can be expressed in E. coli while still maintaining functionality. The assay was carried out using purified FDH protein. Soluble protein fractions from FDH-expressing E.coli and WT E. coli were used as controls.

![](https://static.igem.org/mediawiki/2019/6/63/T--Edinburgh_UG--exp2.png)
Figure 3: 0.1M 6.8pH sodium phosphate, 0.2mM NADH, 0.1M sodium bicarbonate and 0.2µM FDH were mixed together, and the absorbance was measured at 340nm. 0.5mg/ml of protein from soluble fractions of controls were added to the reaction mixture described above. All measurements were done in triplicates and averaged.

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