Team:Groningen/Project

From 2009.igem.org

Abstract

Heavy metal Scavengers with a vertical gas drive.

Introduction

Human health and the environment are endangered by heavy metal pollution in water and sediment. To improve purification strategies a metal selective microbacterial cleaning system was designed. The system comprises uptake, sequestering and metal sensitive buoyancy. All subsystems are interchangeable, which makes it suitable for almost any metal cleaning assay. For this project the modular system was focused on arsenic accumulation, using Escherichia coli as a chassis organism. Arsenite and arsenate are imported by GlpF, a aquaglycerol porin from E. coli. Intracellular As(III) and As(V) are sequestered by fMT or ArsR. These proteins were used as the accumulation modules. Since E. coli does not have a buoyancy system, the polycistronic gas vesicle protein gene cluster from Bacillus megaterium, GVP, was used. The arsenic promoter from E. coli, pArsR, is regulated by the negative transcriptional regulator ArsR. GVP, under regulation of pArsR, was used as the metal sensitive buoyancy module.

Results

All modules were cloned according to the BioBrick^TM Standard Assembly (RFC 10). The synthetic gene GlpF , was successfully cloned into a synthetic operon, with fMT. The GVP cluster, with a ten times repeat sequence, was successfully cloned downstream of the pArsR promoter. These two subsystems were transformed in E. coli to complete the system. The system and its subparts were tested using several assays. Accumulation was tested by an uptake assay, however, since no reproducible results were obtained, the functionality of the accumulation module could not be determined from these data. Arsenic uptake was examined using a metal sensitivity assay. The E. coli strain overexpressing GlpF showed a decreased final cell density upon induction with As(III), suggesting functional expression of the transporter. The metal sensitive promoter pArsR was tested using a fluorescence assay. This showed a 2.3 fold increased activity upon induction with 100 µM NaAsO2. Buoyancy was tested by a sedimentation assay. Enhanced buoyancy was shown for the buoyancy module and the complete system, though no difference of the buoyancy phenotype could be observed upon addition of the accumulation module. Cells cultivated in aerobic conditions showed improved buoyancy compared to cells cultivated in semi-aerobic conditions. An interactive computer model was made for the whole system, with which the modules were further characterized. With the model, import rates of As(III) at different initial extracellular arsenic concentrations could be determined. Also the influence of different parameters on the accumulation factor, the ratio between bound and unbound arsenic, was calculated. The model also allowed qualitative determination of the regulation of pArsR by various expression levels of ArsR. Furthermore, the volume fraction gas vesicles in the cells needed for buoyancy, for several sizes of the gas vesicles, was computed.

Conclusion

The metal selective microbacterial cleaning system for arsenic was shown to be buoyant and the buoyancy module and uptake module were shown to work individually. For a better determination of the system an accumulation assay need to be redone. It was shown here that the system has potential as a cleaning system for arsenic. As mentioned earlier this modular system can also be implemented in cleaning of other substances. Literature research showed possible modules for copper, zinc, mercury and even gold. So not only cleaning water and sludge but also mining rare metals could be functionalized using this system.

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