Team:IISER-Pune-India/TeamNotebook/Team Notebook 11e9906b405543a29361e38a50c81024/Wet Lab Meeting - 6 with PhD Mentors 2a86db39feff4e20849d6146f3a74369

Wet Lab Meeting - 6 with PhD Mentors

Wet Lab Meeting - 6 with PhD Mentors

Date
DepartmentWet Lab
DescriptionWet lab updates and planning
HP sub-branch
Links/media
Participants
Property
Property 1
Property 2
TypeMeetings
  • 8 glycerol stocks each have been made for KJK01 and MG∆pafl and is being stored at -80C at saikrishnan's lab(?). They arrived in 2 plates and 2 cryo vials. One of the plates, which contained KJK01, arrived broken.
  • More stocks of KJK01 must be made. It must also be made competent to transformation and be tested to ensure it is producing butanol. Butanol production can be tested by HPLC or GC-MS, Yazdani sent protocol for HPLC. We may not have a column specific to butanol to check whether it is being produced. We need to ask around to see what can be done about it - if any columns exist for butanol, and whether we need a column specific to butanol anyways.
  • Ask - Sudha, Sagar, some synthetic organic chemist, jayakannan
  • Sagar's lab identifies metabolites produced by plants en masse so ask him how they do it
  • MG∆pafl can be used as control for Butanol production, also we must confirm with Yazdani if it doesn't produce butanol. It also has many of the knockouts needed for succinate production, along with knockouts of genes essential to succinate production (fumarate gene). If we have time we could consider doing succinate production as well. But rushik says focus on butanol as we have the strain already and we must get cracking!
  • We also must order things asapppp!!! Try to order EVERYTHING by June end
  • Akash and Likhith presented CyanoGate stuff to PhD mentors. Rushik read the protocol and said it would be doable. Protocol would need at least 9 days to do according to Akash. CyanoGate parts available on AddGene. Alternative: empty backbone by Lin, Pakrasi also available on AddGene. The CyanoGate kit builds up on the Plant MoClo kit so we need to read up on that too. We also need to narrow down the various backbones we will be needing for the Level 1 and Level T assemblies. And then we can speak to Prem and Veeral and get their inputs and ask if these parts are available at their lab, and accordingly place orders for UTEX and parts from Wangikar.
  • We also need to place orders for the helper strain used in CyanoGate, the ingredients for BG-11, terrific broth, lights, cscABK, sps genes, other standard parts from the registry etc
  • Ashwin will have to go check out the incubator, check its dimensions, feasibility to fix lights and wires, take pictures of incubator (to show prem and veeral) and lab (for the safety form).
  • Rushik also says that having an IPTG inducible promoter for E coli makes sense because you want to control how much gene product is being produced because too much might be toxic to the E coli. He suggested 4 plasmids that they commonly use in their lab:
    • pRSF - kanR
    • pHIS17 - ampR, arabinose induction
    • pET15B - ampR, IPTG induction
    • pET21D - ampR, rushik doesnt remember what it is induced by