Meeting with Rushik - 2
| Date | |
|---|---|
| Department | Wet Lab |
| Description | Queries on getting started with wet lab work |
| HP sub-branch | |
| Links/media | |
| Participants |  Misaal Bedi AKASH DUTTA |
| Property | |
| Property 1 | |
| Property 2 | |
| Type | Meetings |
MG1655 is very easy to grow anywhere, it is not a specialist, and can live on a wide range of conditions, as opposed to strains like BL21(DE3) which may not live if the pH is changed even slightly
KEGG - we can look at the existing knockouts present in Yazdani's strain to see their functions and if it might affect any pathways we need
HB101 is not available in IISER and we would have to order it, we could ask for other similar conjugation strains used in IISER
Cloning methods - RF cloning is the easiest
Clone > Transform > Clone Checking by sequencing
We can check if the bacteria have been transformed with plasmid using antibiotics, but we also need to check if the plasmid contains our gene of interest.
IISER does sequencing by sending samples to someone in Bangalore, takes a week maybe, we need to tell them what primers we have used, so that they can do sequencing
Get strain (butanol producing, cyanobacteria) + Media > Look at Knockout info > grow the bacteria in media (do some experiments in different media compositions, like whether e coli grows in the presence of bicarbonate, pH, with and without sucrose in the BG-11 medium) > Optimise growth conditions for cyano and e coli > then get genes and start cloning
Get gene sequences, see if they have stop codons if you want to put them under same promoter, get sequences of plasmid you want to clone it into, see if you want to use standard plasmids already containing promoters and terminators (e.g. T7) or if you want your own promoters, dont try to replace promoter in a standard plasmid, find plasmid that already has this promoter, or maybe use empty plasmid? see if you can use the plasmid that the gene comes in when you buy it
"We can send you the butanol producing strain. I think E. coli KJK01 will be an ideal strain to work with as it is very easy to handle, can grow in simple media like LB or Terrific Broth, and will produce butanol along with pyruvate which is also a valuable product. I am copying to Dr. Brajesh Barse, who can send you the Material Transfer Agreement form, which you can get it signed by your institute, following which we can dispatch you the strain.
E. coli KJK01 is a facultative anaerobe, you do not need any glove box or CO2 incubator. You can simply grow in shake flask with 50% of the volume filled (e.g., 50 ml culture in 100 ml flask). You can also grow them in a 15 ml of 50 ml Falcon with 2/3rd volume filled. All this will make micro-aerobic culture, which will be sufficient for butanol production. Any DNA transformation can be done just like you do for any lab E. coli cells, like DH5a. However, if you want to prepare the construct by cloning, ligation, etc, it is better to use DH5a due its high transformation efficiency."
These are standard E coli culture conditions!!
Create a schematic for promoters, genes etc we want to use in a plasmid
Keep record of these plasmid maps, gene and primer sequences etc!!!
@AKASH DUTTA @Misaal Bedi add any stuff I may have missed