Team:Groningen/Project/Lab plan

From 2009.igem.org

(Difference between revisions)
(New page: ='''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 accumul...)
Line 41: Line 41:
*BioBrick (BBa_K129004) LamB + metal binding domain is not available
*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
*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
 +
*Start ordering materials like: media, restriction enzymes, kits
 +
*Talk with Profs for permission to use stuff from the different groups in Haren

Revision as of 20:21, 12 June 2009

Contents

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 restrictionsites.
  • 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
  • Start ordering materials like: media, restriction enzymes, kits
  • Talk with Profs for permission to use stuff from the different groups in Haren
Retrieved from "http://2009.igem.org/Team:Groningen/Project/Lab_plan"