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Team:Groningen/Project
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## The metal transporter and accumulation protein should be cloned in parallel. | ## The metal transporter and accumulation protein should be cloned in parallel. | ||
##Then clone them in one vector (in parallel for different systems for Cu, Zn, As, (Hg)). If possible on a synthetic operon. | ##Then clone them in one vector (in parallel for different systems for Cu, Zn, As, (Hg)). If possible on a synthetic operon. | ||
| - | # PCR the restriction sites out and add BioBrick pre- and suffix. | + | # PCR the restriction sites out and add BioBrick pre- and suffix --> Use [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10|BBFRCF10]. |
##Primers should be ordered for HtmA | ##Primers should be ordered for HtmA | ||
# Add a RBS (BBa_B0034) and a terminator (BBa_B0014). | # Add a RBS (BBa_B0034) and a terminator (BBa_B0014). | ||
Revision as of 15:01, 24 June 2009
 | The Project
|
|---|
For our team
| 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 |
Introduction
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.
Order of action:
- Buoyancy
- Metal importation
- Accumulation
- Metal sensitive promotor
Basic Cloning Strategy:
- Transform E. coli TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins.
- Test expression / phenotype of separate proteins (if possible in the vectors they are supplied in).
- Put both systems (gvp and metal import) on a high and low copy number to prevent that the plasmid / expression of one of the systems is not compatible with the other. E.g. use pSB3K3 with p15A, Kan resistance and pSB1AC3 containing Amp + Cam and a pMB1 ori.
- The metal transporter and accumulation protein should be cloned in parallel.
- Then clone them in one vector (in parallel for different systems for Cu, Zn, As, (Hg)). If possible on a synthetic operon.
- PCR the restriction sites out and add BioBrick pre- and suffix --> Use [2].
- Primers should be ordered for HtmA
- Add a RBS (BBa_B0034) and a terminator (BBa_B0014).
- For gvp the RBS is included in the construct.
- In parallel clone metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
- In parallel clone different promoters (inducible like Para or Plac, constitutive with expected high and low expression yield) in front of the two systems.
- Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors.
