Team:Victoria Wellington/Notebook

Notebook

Meeting Minutes

Week 1 - April 23rd

The Postgraduate students in the team started looking for funding and began thinking about possible ideas for the project.

Week 4 - May 13th

The Undergraduate portion of the team was finalised and all members began meeting weekly to discuss and research potential projects. We each came up with a potential project and presented it to the group. After the first round of ideas, we paired up and honed in on projects that looked promising. Finally, we settled on tropine synthesis being the most plausible and exciting project for us. We also investigated potential collaboration sources.

Week 7 - June 3rd

This was our first meeting once we had decided on our project and were discussing how to begin working towards our goal. The first thing we worked on involved organising sponsorships from Geneious and NEB. We decided on team leaders and started discussing the promotional video development. Up until this point we hadn’t done any work in the laboratory, but started ordering parts we needed. We began working on grant applications.

Week 9 - June 17th

We spent time researching Escherichia coli and cyanobacteria growth conditions, expression conditions, expression vectors and characterised promoters. Researching relevant literature to design our project.

Week 10 - June 24th

Went spent time this week working on the Impact Grant Application as well as our promotional video. At this stage we had determined the pathway for tropine synthesis and identified all the genes required for our genetic engineering. We assigned the different projects to people and began meeting in our groups to work on our sections of the project. In the lab we made BG-11 media.

Week 11 - July 1st

We discussed the genes we needed to order and the assembly standards for the parts to be submitted to the competition. We ordered plasmids from IDT for the Synechococcus elongatus cloning. The first part of the lab work for the project involved doing our gene synthesis for E. coli.

Week 12 - July 8th

We met with the Madrid team and discussed potential collaboration. We did our lab inductions this week.

Week 13 - July 15th

We did our PC2 training this week. We filmed our promotional video which you can see below.

Week 14 - July 22nd

We ordered vectors from Addgene. Plasmids are ordered. The Synechococcus elongatus UTEX2973 strain arrived and so the Synechococcus growth team was able to start in the lab. We discussed a presentation for the SBA conference. We met with the Marburg/Bielefeld team.

Week 15 - July 29th

Created the team twitter account.

Week 16 - August 5th

In the lab this week we set up some UTEX2973 cultures and made Electrocompetent cells.

Week 17 - August 12th

In the lab this week we tested the electrocompetent cells, Made BG-11 media and worked to improving growing the UTEX2973 strain. We ordered the primers from IDT.

Week 18 - August 19th

We went into a snap lockdown so we had to halt all our lab work, this massively impeded our progress and ultimately resulted in us moving to a two year project. We presented our project at the Twist Bioscience Discovery event. We decided to use the Impact Grant for the Jamboree registration fee. The plasmids from Addgene arrived. We ordered PCC 7942 & 11801 from the Pasteur Collection.

Week 19 and 20

We discussed the implications of the changing alert levels. We spent this time planning what we could do when we were able to get back into the lab. Planned to set up the cloning with the plasmids and testing of the competent cells when we could get back into the lab: Plan to streak out UTEX2973 onto BG-11 agar, test competent cells, make competent cells for MC1061 strain and do more modelling on AlphaFold for missing PKS enzyme as well as some rational protein engineering (attaching solubility tags, decorating with hydrophilic residues, BASILIScan tool).

Week 21 - September 9th

Discussed the bio bricks and the progress on the wiki. Discussed changing to a 2 year project due to the effect of the snap lockdown The undergraduate students were still not able to get into the lab at this point due to the lockdown. We discussed the potential for a Florida collaboration All miniprepping of the plasmids (the 2 triparental mating plasmids and 4 cloning plasmids) had been completed by this point and we tested the electrocompetent ability of the cells this week (electrocompetent HB101). The E.coli cloning strains were streaked out and glycerol stocks were made. We had trouble growing the cyanobacteria strain as we tried to optimise the growth conditions.

Week 22 - September 16th

We made and tested chemically competent E. coli cells this week and made the triparental mating strains. The vessels we required for bioreactors finally arrived. The Cloning team began Gibson Assembly cloning this week after the plasmids were prepped last week. We confirmed the miniprepping of the plasmids was successful through restriction digestion.

Week 23 - September 23rd

Focused on completing the deadlines that were due this week. We completed the remainder of the Gibson cloning to assemble the ordered gene parts into the expression plasmids. Focused on making the plasmids and getting them into their respective organisms. We began screening the plasmids. Discussing preparations for beginning the CRISPR part of our project.

Week 24 - September 30th

We began uploading parts to the registry and continued working on the wiki. In the lab this week we made some electrocompetent cells EC100 and did plasmid prep and golden gate assembly.

Week 25 - October 7th

The wiki content was divided up and everyone began working on different sections. In the lab we observed some slight growth of the cyanobacteria. Our results of the miniprep and PCR of the Gibson plasmids showed that the 3 genes weren’t integrated properly therefore we repeated the Gibson assembly cloning with higher concentrations of each component.

Week 26 - October 14th

In the lab this week we had some more promising-looking Minipreps for the Synechococcus elongatus constructs from the latest round of transformations we did. These were PCR-checked using primers for both ligation sites. One ligation site could be confirmed confidently while only faint bands were obtained for the other. PCRs with faint bands will have to be repeated with optimised annealing temperatures.

Week 27 - October 19th

Final wiki push. The results of the latest minipreps meant that we needed to repeat the NEBuilder® HiFi assemblies for the E. coli constructs and potentially clone the NEBuilder® HiFi assemblies into another E. coli expression strain.