Team 2: Minicell production and plasmid control

[Zoepin]

In this issue, team 2 will share their research and protocols for minicell production and plasmid control.

[camilaball]

https://docs.google.com/document/d/1ILyT5GAWkq3NXHXZ177cDpo1ojwDqp6D/edit

In this document there are notes for dual plasmid system and the roles of each min proteins which could help us decide what kind of min mutant we would like to use for our project. There is also a list of the different strains/min mutants past igem groups have used for their minicell project

[Zoepin]

Meeting 05.05 - Camila and Zoé

Choice of a min mutant

We have different options in the choice of minicell producing e.coli. We chose to work with mutants with a deletion of the entire min operon (proteins minC minD minE like the previous iGEM teams did.

Dividing the work in 4 parts

Plasmid construction

• Choice of the parts (promoter, RBS, backbone + GFP)
• Design and amplification + design of primers for PCR verification
• Assembly We are thinking of using Golden Gate Assembly, MoClo.
• Transformation and verification

We would like to have a meeting with Aya to confirm our choices and ask our many questions.

Mother cell/Minicell characterization

• Separation of mother cell and minicell => antibiotic method?
• Yield of minicell production

Presence of the plasmid in the minicell

Hypothesis: if the plasmid copy number is high enough, there should be some present in the minicell once it is produced. We want to test this hypothesis:

• Determine plasmid copy number in mother cell
• Determine plasmid copy number in minicell

If the hypothesis is confirmed, we determine the yield of GFP in the minicell.

Once these experiments are prepared, we will prepare experiments in the case our hypothesis is not verified. We will look into plasmid partitioning systems. This solution being more complexe, we would like to avoid it if possible.

Leakiness of the plasmid

If we manage the 3 previous steps, we would like to measure the leakiness of the plasmid, its activation in an environment where it should be inactivated.

[camilaball]

Meeting 07.05 - Zoé Aya and Camila

This meeting was a question session where Zoé and I asked Aya some questions regarding our part of the project.

Question 1: What would be the best approach for our system: having a dual plasmid system where one plasmid contains a repressor + partitioning system or having a plasmid activated by environmental factor?

• Dual plasmid system: The Problem is that plasmids could end up fighting for the genome machinery for replication which could have an impact on the number of plasmids in the end. To avoid that we would have to be careful when choosing plasmid backbones, we would have to be really careful that the two plasmids don't contain the same origin of replication.
• Environmental cue activation: This would be difficult to implement as this would make the design quite dependent on the hardware team's design. For example, if we were to make the plasmid only activated when there is the presence of arabinose this could only be implemented if the device would spit out minicells and they would be put in a different location from the mother cells. However, if the minicells are separated from the mother cells by flow this would be hard to implement.
• Integrating the repressor inside the genomic DNA: What seems like the better option now would be to use either CRISPR or P1 transduction to integrate a repressor for the plasmid that way once the minicells will be formed there will not be any more production of the protein. However, there will be probably be a latency period between minicell formation and expression of the gene of interest as even once the minicells will be formed the repressor protein that was produced in the mother cell will be present. Therefore, we will need to add a degradation tag to increase the degradation of the repressor.

Question 2: To maximize our yield, do we need to have a strong promoter + strong RBS + High Copy Plasmid? Or would that be too much and be too much burden?

• Our choice of promoter depends on our repressor, therefore we are already quite limited on our choices of promoters. However, if we end up having to choose a weak promoter we will need to have a strong RBS.
• High copy plasmid is the best option

Question 3: We will do proof of concept with GFP, will the system behave differently if we add a different protein to GFP as a fusion protein?

• Need to test with GFP always so that we can understand the mechanism of the system
• We will need to have 2 reporters: one for the repressor (RFP for example). Once RFP will be degraded we will see how long the actual repressor will take to be degraded.

Question 4: How do we choose the backbone? ColE1?

• High Copy plasmid

Question 5: Where do we integrate into the bacterial genome ?

• Need to look into protocols and papers. Use nutritional markers?

For next week:

• Need to look for repressor +promoter
• Where to integrate into the genome
• P1 transduction and CRISPR
• Degradation tag