Revision as of 12:40, 15 June 2009 by Nienke (Talk | contribs)

Lab plan:

Clone gvp from BioBrick (BBa_I750016) in vector with inducible promoter (e.g. PAra)
Clone a metal ion transporter (CitM and HtmA are planned)
Clone a metal accumulating protein.
Clone a metal sensitive promoter in front of the gvp cluster.
Clone the metal accumulating protein and metal transporter in one vector, possibly on a synthetic operon
Transform both vectors in one E. coli strain

Gvp Cluster

BBa_I750016 is a construct: E-genR-X-RBS-PART-S-P in vector BBa_J61035.
Possibly the AmpR and ColE1 are also still present on the vector, as the part was last checked in 2008 and contained a Amp resistance marker…
The part is present in the microtiter plate: Kit Plate 2, Well 11A
Quality control was okay, but plasmid length was different than expected. --> should be checked by us.
Plan:
- Get chemically competent E. coli
- Dilute dry DNA of gvp with 15ul diH2O
- Transform to E. coli --> see protocol
- Plate on LB-A plates with Amp or Gentamicin selection (50/50)
- Use positive and negative control
- Grow o/n @ 37 degrees
- Pick single colony and grow o/n in 5ml LB + correct antibiotic
  - Make glycerol stocks of the o/n culture
  - Do DNA isolation
  - Check the vectors insert by restriction analysis --> PstI and XbaI (would give 1 band of ~ 3539bp and 1 of 6064bp)
Design primers to get rid of restrictionsites

SJ transformed E. coli with pWSK29, a low copy nr vector containing CitM (according to B. Krom this is the best vector for CitM)
Get plasmid and possibly the plate with E. coli transformants.
- Check expression of CitM while growing on citrate medium with Amp, induce with IPTG
Check gene for restriction sites and design primers to get rid of restriction sites.
Design primers to introduce a pre/suffix --> ask B. Krom whether this would be feasible

BioBrick (BBa_K129004) LamB + metal binding domain is not available
Find out whether it’s possible to only express the metal binding domain (6AA) and if so, order synthetic DNA with part with pre/suffix

Get CitM from the biological centre
Finish equipment list --> send to facility manager
Design primers
Read articles to find modeling parameters for the different sub-projects and to find testing-phenotype (e.g. metal inport) protocols
Start ordering materials like: media, restriction enzymes, kits
Talk with Profs for permission to use stuff from the different groups in Haren
Check compatibility of different plasmids / promoters / replicons in E. coli--> On Fermentas site about pUC18, pUC19 and about pACYC184