From 2009.igem.org

Lab plan:

1. Clone gvp from BioBrick (BBa_I750016) in vector with inducible promoter (e.g. PAra)
2. Clone a metal ion transporter (CitM and HtmA are planned)
3. Clone a metal accumulating protein.
4. Clone a metal sensitive promoter in front of the gvp cluster.
5. Clone the metal accumulating protein and metal transporter in one vector, possibly on a synthetic operon
6. 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

CitM

• 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

HtmA

• On its way, but hopefully arrives next week

Accumulation of metal

• 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

To do list

• 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 [http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm pUC18, pUC19] and about [http://www.fermentas.com/techinfo/nucleicacids/mappacyc184.htm pACYC184]