Team:Groningen/Project

From 2009.igem.org

Revision as of 10:17, 14 October 2009

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:

As a result of our project, we hope to acquire the following 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:

We have been carrying out our tests in the following order:

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

Basic Cloning Strategy:

We clone the organisms using the following 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

Teams with similar projects

→ UQ Australia

Water contamination is a key environmental issue for many countries around the world, both developed and developing. In Queensland, Australia we have a particular problem with Mercury (Hg2+) contamination of water supplies around the major mining town of Mt Isa. After searching through the iGEM projects from previous years, the arsenic detection system inspired us. As the UQ 09' team we wish to take this idea one step further and completely remove Mercury from water systems.

To do this we will be utilizing a strain of Escherichia coli, and the already established mercury uptake, reduction and efflux system and making a few modifications. One of our aims is to couple the detection of Mercury to the expression of a native bacterial protein, Antigen 43 (AG43). This protein, when expressed, causes the bacteria to stick to one another. As the bacteria aggregate in clumps, they will fall to the bottom of the sample. Our idea is for the bacteria to take up the mercury, activating Ag43 expression, resulting in aggregation and the Mercury-filled bacteria will fall to the bottom leaving clean water.

There are a number of parts that we hope to add to the registry. The first is Ag43 as a protein coding sequence and the MerR promoter sequence. We will also add the completed mercury uptake and aggregation system as an operon.

→ Newcastle

The aim of our project is to genetically engineer Bacillus subtilis to be able to detect and sense cadmium which has been taken up from the soil environment and sequester them into a metallothionein. This metallothionein will then become incorporated into a Bacillus spore; the resilience of which means that the cadmium ions can become isolated from the environment (and made bio-unavailable) for many years.

This project involves a number of steps, each of which can be considered as sub projects:

1. Metal intake
2. Metal sensing
3. Tuning of Bacillus subtilis normal stochastic switch
4. Metal sequestration by metallothionein
5. Second stochastic switch
6. Synthesizing a Promoter Library for Bacillus subtilis

For our team