Meeting with Prem and Veeral - 6
| Date | |
|---|---|
| Department | Wet Lab |
| Description | Finalising list of parts, Pakrasi strain updates |
| HP sub-branch | |
| Links/media | |
| Participants | |
| Property | |
| Property 1 | |
| Property 2 | |
| Type | Meetings |
Final List of Parts submitted to Prem and Veeral:
Strains:
1. Synechococcus elongatus UTEX 2973 WT
2. Synechococcus elongatus PCC 7942 WT (as back up as Prem had suggested)
3. E coli HB101 (with plasmids pRL443 and pRL623 - conjugal and helper plasmids respectively)
Plasmids:
1. pSL2680
2. pSyn_1 or pSyn_6 (as a backup)
Promoters:
1. Pcpc560 (available in the CyanoGate collection - pC0.005) - strong promoter native to Synechocystis PCC 6803
2. Bba_J23119 (available in the CyanoGate collection - pC0.049, might be available at IISER too; need to cross-check) - strongest promoter
3. J23119 V02Â (available in CyanoGate collection - pC0.084) - weaker promoter
4. PcpcB m6 (characterized in A. Sengupta et al., 2020) - new strong promoter we could try to characterize, mutant version of PcpcB from PCC 7942
5. PpsbA3 (used in Wangikar et al., 2020, characterized for UTEX in Shubin Li et al., 2018) - strong promoter native to PCC 7942 and UTEX 2973
Terminators:
TrbcL or Trrnb or Tcpc (the last two are available in the CyanoGate collection)
Restriction enzymes:
Kpn1 or Sal1 - restriction sites on pSL2680
Aar1
Antibiotics:
Kan, Spec, Amp
List is okay
But they can't provide us with parts from CyanoGate due to the agreement under which they would have obtained it. So J23119, and J23119 V02 cant be provided to us from CyanoGate collection. However, J23119 might be available in the 2019 distribution kit. They can provide us cpc560 in a plasmid, and not from the CyanoGate collection.
They can't provide us with REs, as they are expensive and they have only small quantities in their lab. We will have to obtain it ourselves. We could buy it from Thermo Fisher.
Lighting: They use 14 Syska tube lights and are able to achieve a light intensity of 400-450 micromoles with it
Cpf1 won't contain restriction sites for Kpn1, Sal1 or Aar1, but even if it does, there are methods to introduce a point mutation in order to remove the restriction sites.
Repair template design:
Kpn1 site - 750bp UF homology arm - Restriction site for RE of choice like Nde1 - promoter - Nde1 site - cscB gene - terminator - 750 bp DF homology arm - Kpn1 site
Overlap PCR or CPEC for joining the parts together, restriction ligation for repair template into pSL2680 backbone, and replacing promoters
Kpn1 or Sal1 site can be used to clone in the repair template. Nde1 is useful because the recognition site contains ATG - which would be start codon for cscB gene
Aar1 site would be used to clone in the gRNA
We must check all parts for for the presence of restriction sites for the enzymes we are using. Check for PAM site in all our parts as well, it shouldn't be present. Check homology % b/w 7942 and UTEX, or we can amplify the arms directly from the WT UTEX.
We can synthesise PAM site? Synthesise for what??
gRNA design:
20 bp - PAM - Aar1 site
All the assembly needs to be done in an intermediate plasmid like pSyn_1 or pSyn_6 and then pasted into pSL2680
Receiving strains from Pakrasi:
Ask thomas what all GMO shipping paperwork we need to get ready, talk to someone who has gotten this tuff shipped before and what all goes into it. Confirm with Michelle if there is any additional paper work/documentation needed from you.