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Team:Groningen/Project
From 2009.igem.org
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==Basic Cloning Strategy:== | ==Basic Cloning Strategy:== | ||
# Transform ''E. coli'' TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins. | # Transform ''E. coli'' TOP10 (genotype DH 10B) with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins. | ||
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# PCR the restriction sites out and add BioBrick pre- and suffix --> Use [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10 BBFRCF10]. | # 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 | + | ##Primers should be ordered for the different genes. |
| - | # Add a RBS (BBa_B0034) | + | ## Add a RBS (BBa_B0034)in the primer for the BioBrick prefix. |
| - | ## For gvp the RBS is included in the construct. | + | ## Add a terminator (BBa_B0014) via cloning. |
| - | # In parallel clone metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster. | + | ## For gvp the RBS is included in the construct, and biobrick suffix is included in the construct. The prefix is missing because of the ''Eco''RI site in the middle of the plasmid!! This may give problems!! |
| - | # In parallel clone different promoters | + | ## 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 (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain. | ||
| + | ## The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon. | ||
| + | ## Clone the different systems for Cu, Zn, As, (Hg) in [http://www.partsregistry.org/Assembly:Rolling_assembly parallel]. | ||
| + | # ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster. | ||
| + | # ''(If needed and not already supplied by "vector group")'' [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] different promoters in front of the two systems. | ||
| + | ## Inducible like Para or Plac | ||
| + | ## Constitutive with expected high and low expression yield | ||
| + | ## Metal sentitive promoter (only for gvp system) | ||
# Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors. | # Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors. | ||
Revision as of 21:54, 2 July 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
| 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.
- 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 the different genes.
- Add a RBS (BBa_B0034)in the primer for the BioBrick prefix.
- Add a terminator (BBa_B0014) via cloning.
- 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!!
- 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 (supplied by "vector group"). This is needed to prevent plasmid / expression incompatibility when both systems are used in one strain.
- The metal transporter and accumulation protein should be cloned behind each other. If possible on a synthetic operon.
- Clone the different systems for Cu, Zn, As, (Hg) in [http://www.partsregistry.org/Assembly:Rolling_assembly parallel].
- (If needed and not already supplied by "vector group") [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
- (If needed and not already supplied by "vector group") [http://www.partsregistry.org/Assembly:Rolling_assembly In parallel clone] different promoters in front of the two systems.
- Inducible like Para or Plac
- Constitutive with expected high and low expression yield
- Metal sentitive promoter (only for gvp system)
- Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors.
