Revision as of 14:50, 10 July 2009 by Verhoeven1981 (Talk | contribs)

The Project

For our team

Missing / available information
Metal Transporter Inducible promoter Regulator Accumulation protein
Arsenic GlpF (organism?) - ordered Promoter region of ?? gene, responding on ArsR ArsR (E. coli)- ordered ArsR
Copper HmtA (Pseudomonas sp.)- available None found in E. coli Idem ??
Zinc HmtA (Pseudomonas sp.)- available ?? ?? ??
Mercury MerT (E. coli and other sp)- PCR? Should be available in E. coli - PCR? Idem Idem


For the lab work of our project, concerning the bouyant bacteria with its metal absorbing and accumulating "function", we hope to produce the following products by using our basic cloning strategy.

Final products:

  • Plasmid with gvp cluster, regulated by different promoters. [link to BioBricks]
  • (Several) plasmid(s) with a metal transporter, a metal accumulating protein and if needed a regulator protein for the metal sensitive promoter. [link to BioBricks]
  • One E. coli strain with both systems.

Part List for our -80 freezer!

Order of action:

  1. Buoyancy
  2. Metal importation
  3. Accumulation
  4. Metal sensitive promotor

Basic Cloning Strategy:

  1. Transform E. coli TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins.
  2. PCR the restriction sites out and add BioBrick pre- and suffix --> Use BBFRCF10.
    1. Primers should be ordered for the different genes.
    2. Add a RBS (BBa_B0034)in the primer for the BioBrick prefix.
    3. Add a terminator (BBa_B0014) via cloning.
    4. For gvp the RBS is included in the construct, and biobrick suffix is included in the construct. The prefix is missing because of the EcoRI site in the middle of the plasmid!! This may give problems!!
  3. PCR restriction sites out. !!Both PCR reactions for pre/suffix and restriction sites can possibly be done in 3 PCR reactions --> Ask Frans or J. Kok!!
  4. Test expression / phenotype of separate proteins (if possible in the vectors they are supplied in).
  5. Put both systems (gvp and metal import) on a high and low copy number (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain.
    1. The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon.
    2. Clone the different systems for Cu, Zn, As, (Hg) in parallel.
  6. (If needed and not already supplied by "vector group") In parallel clone metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
  7. (If needed and not already supplied by "vector group") In parallel clone different promoters in front of the two systems.
    1. Inducible like Para or Plac
    2. Constitutive with expected high and low expression yield
    3. Metal sentitive promoter (only for gvp system)
  8. Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors

Quality control

What to do after transforming your cells with a vector:

  • (Grow o/n culture as usual
  • Streak with "entoog" a little out for single colonies.
  • Thereafter you can continue to miniprep the cells and check insert length by PCR/restriction analysis (Quality control --> Overdrachts document iGEM 2008).
    • For restriction analysis, do a double digest, and try to find a restriction enzyme which only cuts in the vector and one which only cuts in the insert...
  • Pick one (very) single colony, try hard not to pick a satellite colony!
  • Grow o/n culture and make glycerol stock (keep track of were you put them in "constructs" file on Google Docs!) next day.
  • Miniprep and continue with cloning / checking insert length.

Tracking information about cloning

  • Constructs / plasmids lokation in GoogleDocs document called: Constructs.
  • Primer / Oligos in GoogleDocs documents called: Primers.
  • What we take from MolGen stocks in a GoogleDocs document called: Chemicals / Materials taken from MolGen.
    • This list was also put on the door in the lab.
Part List for our -80 freezer!