- Do we order everything at once? Order now, it'll take time to come.
- How long will the things from Wangikar's lab take to reach Pune? Not much time lol [10-12 days before sending]
- BG-11 we'll get general composition, we can make it ourselves also. Buying is preferred if we don't have some ingredients. (20-30L)
- They'll give the protocols [Important!!]
- Order filter paper for shifting stuff from one antibiotic to the other while doing conjugation and transformation
- Natural transformation (PCC 11801) - stuff we need - Agar BG11 plate with low antibiotic. 10ml low (0.7) OD culture, antibiotic plate. And you have to gradually increase antibiotic. Segregation takes time. Check for segregation using PCR.
- Take normal LED bulb (monochromatic is better)
- CO2 supply - Bicarbonate (60mM), CO2 controlled chamber CO2 conc.- ambient for transformations / 1% usually for culturing for product synthesis (but depends on the product)
- Veeral: Bicarbonate may be toxic to E coli cells (won't affect pH that much tho) and might be toxic to cyanobacteria if in excess. Bicarbonate dissolves a lot more than CO2 into medium? So we need to be careful about how much bicarbonate we add. We would have to add small quantities of it every 6-8 hours probably. We also need to acclimate the cyanobacteria to the CO2 levels or bicarbonate levels instead of just dumping them into new conditions.
But Nishad said that E coli wont be affected too much by swings in pH or by bicarbonate presence Ref: Pakrasi's paper on using bicarbonate to cyanobacteria cultures
- Conjugation: stuff we need- BG11 agar plate with 5%LB,
Helper cell - conjugal strain E coli HB101 with pRL443 and pRL623 plasmids - it usually has ampicillin resistance,
E coli with construct - DH5a, 11801
We can get the helper strain from their lab
We need to wash the E coli several times with autoclaved water or PBS to remove antibiotics
We start off with high antiobiotics for conjugation. For PCC 11801, you can transfer to plate directly, for 7942 you can incubate over night.
- Make two connections for LEDs. For high light, use both, for low light, use one. There must be a space to take wires from inside incubator to outside. We can stick LEDs using double sided tape. Prem has even used those tubelights which you can increase brightness using a regulator (like that of a fan). She can send pics to show her set up.
- How to preserve the strain? Store at -80 C. Plates can be kept at 4 C. BG-11 can be kept at room temperature or stored at 4 C but bring it to room temp before using.
- Strain - UTEX 2973, Conjugal Strain/plasmids - E coli HB101 / pRL443, pRL623: From Prof. Wangikar
- Plasmids are available specific to the strain we want to use. They contain the homology arms already for recombination into neutral sites. 7942 plasmids could be used for 2973 as they are very similar genetically, we can use a BLAST search to decide if we can use plasmids from other strains.
These plasmids should be available on addgene
Operon set ups wouldnt work well in cyanoabcteria and so we would have to opt for separate promoters for each gene we want to overexpress.
We could try overexpressing a reporter gene first to see if cloning/transformation method is working.
Read Sam Brooke's handbook on molecular biology - it will contain all the info needed on wet lab techniques needed