iGEM 2021 — Team Victoria_Wellington

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Victoria Wellington/Notebook

[![](https://static.igem.org/mediawiki/2021/4/43/T--Victoria_Wellington--vic_gem.png)](https://2021.igem.org/Team%3AVictoria_Wellington)

Team

[Members](https://2021.igem.org/Team%3AVictoria_Wellington/Team)
[Collaborations](https://2021.igem.org/Team%3AVictoria_Wellington/Collaborations)
[Attributions](https://2021.igem.org/Team%3AVictoria_Wellington/Attributions)

Project

[Description](https://2021.igem.org/Team%3AVictoria_Wellington/Description)
[Results](https://2021.igem.org/Team%3AVictoria_Wellington/Results)
[Challenges](https://2021.igem.org/Team%3AVictoria_Wellington/Challenges)
[Parts](https://2021.igem.org/Team%3AVictoria_Wellington/Parts)

Lab

[Safety](https://2021.igem.org/Team%3AVictoria_Wellington/Safety)
[Notebook](https://2021.igem.org/Team%3AVictoria_Wellington/Notebook)
[Protocols](https://2021.igem.org/Team%3AVictoria_Wellington/Protocols)

About

[Contact us!](https://2021.igem.org/Team%3AVictoria_Wellington/Contact)
[Sponsorship](https://2021.igem.org/Team%3AVictoria_Wellington/Sponsorship)

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.

![](https://static.igem.org/mediawiki/2021/6/69/T--Victoria_Wellington--geneious_logo.png)
![](https://static.igem.org/mediawiki/2021/d/db/T--Victoria_Wellington--twist-logo.png)
![](https://static.igem.org/mediawiki/2021/c/c3/T--Victoria_Wellington--neb-logo.png)
![](https://static.igem.org/mediawiki/2021/c/c0/T--Victoria_Wellington--idt-logo.png)
![](https://static.igem.org/mediawiki/2021/1/1d/T--Victoria_Wellington--asana-logo.png)

![](https://static.igem.org/mediawiki/2021/3/32/T--Victoria_Wellington--VUW-Logo.png)

![](https://static.igem.org/mediawiki/2021/5/5f/T--Victoria_Wellington--science-faculty.png)

![](https://static.igem.org/mediawiki/2021/1/1c/T--Victoria_Wellington--vuw-fe-logo.png)

![](https://static.igem.org/mediawiki/2021/1/1c/T--Victoria_Wellington--ferrier-logo.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Victoria Wellington/Engineering

### ★ ALERT!

This page is used by the judges to evaluate your team for the [medal criterion](https://2021.igem.org/Judging/Medals) or  [award listed below](https://2021.igem.org/Judging/Awards).

Delete this box in order to be evaluated for this medal criterion and/or award. See more information at  [Instructions for Pages for awards](https://2021.igem.org/Judging/Pages_for_Awards).

# Engineering Success

### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

Please see the [2021 Medals Page](https://2021.igem.org/Judging/Medals) for more information.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Victoria Wellington/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Victoria Wellington/Results

[![](https://static.igem.org/mediawiki/2021/4/43/T--Victoria_Wellington--vic_gem.png)](https://2021.igem.org/Team%3AVictoria_Wellington)

Team

[Members](https://2021.igem.org/Team%3AVictoria_Wellington/Team)
[Collaborations](https://2021.igem.org/Team%3AVictoria_Wellington/Collaborations)
[Attributions](https://2021.igem.org/Team%3AVictoria_Wellington/Attributions)

Project

[Description](https://2021.igem.org/Team%3AVictoria_Wellington/Description)
[Results](https://2021.igem.org/Team%3AVictoria_Wellington/Results)
[Challenges](https://2021.igem.org/Team%3AVictoria_Wellington/Challenges)
[Parts](https://2021.igem.org/Team%3AVictoria_Wellington/Parts)

Lab

[Safety](https://2021.igem.org/Team%3AVictoria_Wellington/Safety)
[Notebook](https://2021.igem.org/Team%3AVictoria_Wellington/Notebook)
[Protocols](https://2021.igem.org/Team%3AVictoria_Wellington/Protocols)

About

[Contact us!](https://2021.igem.org/Team%3AVictoria_Wellington/Contact)
[Sponsorship](https://2021.igem.org/Team%3AVictoria_Wellington/Sponsorship)

Results

Cyanobacteria Growth

We did not manage to successfully grow our *S. elongatus* strain UTEX2973. Optimal growth requires BG-11 media, adequate light (400 μmol m-2 s-1) and CO2 and a temperature of 39-41°C. However, over a range of light (50-400 μmol m-2 s-1) and temperature conditions (30-41°C) and BG-11 concentrations no growth was exhibited in either liquid culture or on BG-11 plates. While we did not have the desired control over CO2 concentration we expected growth even at low CO2 concentrations. Ascorbic acid and sodium thiosulfate were trialled as a media supplement in an attempt to eliminate reactive oxygen species (Michael Burgis - personal communication) that can inhibit growth but no effect was observed from this. It is possible our strain was dead due to being held in customs for months in non-optimal conditions.

S. Elongatus Gene Constructs

We had successful transformation of our first *S. elongatus* construct containing genes 1-3 for the tropine biosynthesis pathway into our *E. coli* cloning strain and possible transformation of the second construct (containing genes 4-6). Gibson assembly of the two constructs were carried out and these constructs were then introduced into our electrocompetent *E. coli* cloning strain. These cells were then plated onto antibiotic plates and in each case multiple colonies exhibited antibiotic resistance. Thus indicating successful transformation of the plasmid. Three Colonies were picked for each plasmid and tested via PCR to determine whether our genes were correctly placed in the plasmid. Two PCRs were carried out for each of our constructs with primers that overlapped the region between our introduced gene sets and the backbone plasmids. The setup for the PCR gels is displayed in the table below and the gels are given in the two figures below.

PCR Gel Loading

| S. Elongatus Construct 1, gens 1-3 (figure 1): |
| --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Hyper ladder 1kb | PP1, Se 1-3 colony 1 | PP1, Se 1-3 colony 3 | PP1, Se 1-3 colony 4 | PP1 control | PP2, Se 1-3 colony 1 | PP2, Se 1-3 colony 3 | PP2, Se 1-3 colony 4 | PP2 control |
| S. Elongatus Construct 2, genes 4-6 (figure 2): || 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Hyper ladder 1kb | PP3, Se 4-6 colony 2 | PP3, Se 4-6 colony 3 | PP3, Se 4-6 colony 4 | PP3 control | PP4, Se 4-6 colony 2 | PP4, Se 4-6 colony 3 | PP4, Se 4-6 colony 4 | PP4 control |

Gel Pictures

![Figure 1 - Gibson cloning PCR confirmation gel 1](https://static.igem.org/mediawiki/2021/9/9d/T--Victoria_Wellington--gel1.jpg)

Figure 1 - PCR gel for confirmation of *S. elongatus* gene construct 1 containing genes 1-3. Lane 1 contains 1kb DNA Hyper Ladder, lanes 2-5 contain primer pair 1 and lanes 6-9 contain primer pair 2. Template DNA from the three colonies picked (Se 1-3 1,3 and 4) are present across lanes 2-4 and 6-8 with lanes 5 and 9 acting as negative controls.

![Figure 2 - Gibson cloning PCR confirmation gel 2](https://static.igem.org/mediawiki/2021/b/b8/T--Victoria_Wellington--gel2.jpg)

Figure 2 - PCR gel for confirmation of *S. elongatus* gene construct 2 containing genes 4-6. Lane 1 contains 1kb DNA Hyper Ladder, lanes 2-5 contain primer pair 3 and lanes 6-9 contain primer pair 4. Template DNA from the three colonies picked (Se 4-6 2,3 and 4) are present across lanes 2-4 and 6-8 with lanes 5 and 9 acting as negative controls.

*E. coli Gene Constructs*

We did not manage to successfully transform the two *E. coli* gene constructs into our *E. coli* cloning strain. Gibson assembly was carried out to form both of the plasmid constructs containing genes 1-3 and 4-6 respectively for the tropine biosynthesis pathway in *E. coli*. These were then transformed into our electrocompetent *E. coli* cloning strain however no colonies displayed antibiotic resistance, thus indicating no form of the plasmids (with or without our added gene sets) were successfully introduced to the cells.

Given that the *S. Elongatus* plasmids were successfully transformed into our *E. Coli* cloning strain suggests the difficulty with the E. Coli plasmids were not at the gibson assembly stage. Instead we suggest that since the *E. coli* plasmids contain *E. coli* promoters while the *S. elongatus* plasmids do not, that the metabolic burden on the cloning strain of both expressing the enzymes on the plasmid and replicating the plasmid led to non-viable cells. To combat this in future we could consider having inducible E. Coli promoters on these plasmids, then expression could be inhibited in the cloning strain reducing the metabolic burden of the plasmid and hopefully leading to viable cells for cloning.

![](https://static.igem.org/mediawiki/2021/6/69/T--Victoria_Wellington--geneious_logo.png)
![](https://static.igem.org/mediawiki/2021/d/db/T--Victoria_Wellington--twist-logo.png)
![](https://static.igem.org/mediawiki/2021/c/c3/T--Victoria_Wellington--neb-logo.png)
![](https://static.igem.org/mediawiki/2021/c/c0/T--Victoria_Wellington--idt-logo.png)
![](https://static.igem.org/mediawiki/2021/1/1d/T--Victoria_Wellington--asana-logo.png)

![](https://static.igem.org/mediawiki/2021/3/32/T--Victoria_Wellington--VUW-Logo.png)

![](https://static.igem.org/mediawiki/2021/5/5f/T--Victoria_Wellington--science-faculty.png)

![](https://static.igem.org/mediawiki/2021/1/1c/T--Victoria_Wellington--vuw-fe-logo.png)

![](https://static.igem.org/mediawiki/2021/1/1c/T--Victoria_Wellington--ferrier-logo.png)