Team:Edinburgh UG/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
Sh1 & Associated Maturation Proteins clusters
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.