Team:Groningen/Project

From 2009.igem.org

For our team

Introduction

We hope to produce buoyant bacteria that can filter arsenic out of water/sludge. You can click on the names of the individual parts in the image below to learn more about the different parts of our system.

Final products:

A diagram of the final system in action, showing several different alternatives for filtering metal.

• 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!

In the periodic table below you can see for which elements we have identified a transporter, an accumulating protein and/or promotor. TODO Make list more complete and add links to parts.

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 [http://openwetware.org/wiki/The_BioBricks_Foundation:BBFRFC10 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 [http://www.partsregistry.org/Assembly:Rolling_assembly parallel].
6. (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.
7. (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.
   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