Meeting with Abhinav Masih
| Date | |
|---|---|
| Department | Wet Lab |
| Description | Queries on getting started with wet lab work |
| HP sub-branch | |
| Links/media | |
| Participants | |
| Property | |
| Property 1 | |
| Property 2 | |
| Type | Meetings |
Snapgene - app to help design constructs, they have one month free trials and might sponsor the paid version on one computer
iGEM website has useful info on promoters (whether cosntitutive.induced), RBS, we can take parameter values listed for these parts and give them to Dry Lab. Most commonly used promoters are those induced by IPTG (P lac) and arabinose (P ara)
Promoter choice depends on how much expression you want, we can choose strong or weak terminators, we need to look into the size of the genes and how much distance we want between promoters, genes, terminators etc. whether we want a single promoter for the operon or have different promoters
Biobricks: only sequence needs to be submitted not actual physical copies, has a format - prefix + suffix + in a plasmid
Plasmids must have either kanamycin, chloamphenicol, or ampicillin resistance.
Cloning methods: Using restriction enzymes, Restriction free (RF) cloning using PCR, golden gate, different methods are listed on iGEM website
They spent their first month in May just training, with practise plasmids and not with biobricks. Their wet lab went on till October because they were unsuccessful in transforming.
DH5a is commonly used for cloning as it has a high copy number
another strain is used for expression as it has higher rates of gene expression
Visual assays would be preferred, make sure we have all equipment (spectrometers etc) needed on campus to be able to measure the visual assays if we are doing them.
We can submit both basic and composite parts to the library. For example - the cscA, cscB, cscK operon can be submitted as 6 individual parts including the promoter, RBS, terminator, and the entire operon together can be submitted as a composite part. If we are synthesising these genes we can synthesise it in three parts: promoter + RBS + gene 1, gene 2, gene 3 + terminator - but this would count as 3 parts??
For mentors, we could create a general outline of the sequence of parts in our plasmid design and they can help us with the specific like primers and cloning and all that
For HP - we could do bioreactor design and we could look into mitigating any cyano toxins that might be present along with our final product in our culture to make it safe for use. IHP - developing sensors to check the progress of production or co-culture conditions using biological or chemical sensors in response to feedback from consumers (industries etc)
Primers can be ordered from some place near campus, to reduce work we could have some promoters and terminators synthesized into the backbone but that would reduce our number of basic parts.