Meeting with Rupali
| Date | |
|---|---|
| Department | GeneralWet Lab |
| Description | Presented idea and got feedback |
| HP sub-branch | |
| Links/media | |
| Participants | |
| Property | |
| Property 1 | |
| Property 2 | |
| Type | Meetings |
- Institute has ordered Keio collection containing different E coli strains, each with single knockouts, for all E coli genes (Nishad, Amrita)
- Lamda phage recombineering system can be used to transduce mutations from one mutant to another. This is fairly easy and might take about 3 days to produce one knockout if all goes well. It would be easy to produce double knockouts and they will survive but quadruple knockouts would be difficult and may take 1-2 months,
- CRISPR might take more time to make knockouts as it would take time to design primers and gRNA and order them and then actually carry out the modifications and select for mutants. Generally, these PCR-based modifications (?) might take more time than simply transducing mutations.
- project is wet lab intensive and we need lab space asap and it will take time to order all strains from the US
- You need to look into the antibiotics that you'll be using for cloning and ensure that both E coli and cyanobateria are finally resistant to the same antibiotics? To get around this you could insert genes directly into genome instead of using plasmids?
- Tips:
- create multiple copies of glycerol stock??
- isolate 4-5 colonies from the plate every single time???
- multiple passages? grow over different generations so that favourable mutations might occur?
- For sucrose uptake assays we could look into calorimetric assays. When Akash said that the efflux of sucrose would be equal to influx of sucrose and hence there would be no sucrose in the medium, she said that there would be no way of directly measuring sucrose efflux and influx then.
- Radiometry to analyse sucrose flux would be difficult as it requires considerable training first. The facility on campus only allows dry cultures/slides, and doesn't allow any liquid cultures.
- People to contact:
- FBA: Anu's lab (NCL), Sandeep Krishna (NCBS)
- Modelling in general: Chaitany Athale helped out in 2019, Sutirth for growth rate and pop dynamics
- Wet lab: Nishad and Sunish
- She'll get back to us with whether she can commit to being a PhD mentor for us and that she'll get back to us with possible alternative compounds we can look into. We can always reach out to her for help though.