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Team:UC-Santa Cruz/Project
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Abstract
to save water and make cool bacteria
The world's six billion people are appropriating 54 percent of all the accessible freshwater contained in rivers, lakes and underground aquifers. - See more at: http://www.unwater.org/statistics.html#sthash.JfpKTp6p.dpuf
The total volume of water on Earth is about 1.4 billion km3. The volume of freshwater resources is around 35 million km3, or about 2.5 percent of the total volume. - See more at: http://www.unwater.org/statistics.html#sthash.JfpKTp6p.dpuf
To access this saltwater to accommodate the needs of the global population, many desalination techniques have been developed. The end goal of our project is a bacterial system to directly desalinate seawater using sunlight. The project also aims to provide a scaffold for bacterial manipulation of ionic solutions (ex. heavy metal bioremediation) for other groups.
building a monolayer biofilm to act as a selective membrane. Uses rhodopsins to pump ions in seawater.
Contents |
Project Details
Our project aims to create a microbial desalination system using the bacteria Caulobacter crescentus. This system will use the ion gradients and light to pump sodium and chloride ions out of saltwater. Caulobacter crescentus has two important properties that we hope to use for our project.
First is a special organelle called a stalk. The stalk allows Caulobacter to attach itself to solid surfaces. The stalked cells will accumulate on a surface, and are able to form a monolayer of cells called a biofilm. We will grow these cells on an ion permeable membrane to create a working community of cells that only allow sodium and chloride ions to pass through.
The second property of C. crescentus is the asymmetrical organization of the cell during replication. When C. crescentus replicates, the ends of the cell will localize different proteins to form a stalk on one end, and a flagellum on the other. We will use this property to investigate the potential use of fusion proteins to direct the localization of ion pumps and channels. The result should be a biofilm of cells pumping ions from one side to the other. Theoretically, this will desalinate the seawater on one side, and concentrate sodium chloride on the other.
Part 2
The Experiments
- Transform caulobacter with florescent fusion protein plasmids.
- Induce transformed caulobacter to express florescence.
- Grow monolayer of caulobacter biofilm.
- create tag florescent fusion protien plasmids,transfect into caulobacter, induce, check for polarized florescence.
- create pumps/channels/pores tag fusion protien plasmids,transfect into caulobacter, induce, check for polarized florescence.
- build interface for biofilm (i.e. glass or gel filter)
- Activation of pumps.
Work in Progress
Two plasmids: pvcfpc-4(gentamicin restiant vanillin induced cyan florescence and pxyfpc-2(kanamycin resistant xylose induced florescence)and a wild type strain of Caulobacter (CB15N) were obtained from the group in the paper listed below.
http://nar.oxfordjournals.org/content/35/20/e137.full.pdf+html
Caulobacter was then transfected with each plasmid to create one pvcfpc-4 strain and one pxyfpc-2 strain. Each were then cultured and plated respectively with the antibiotics in which they are resistant.
At OD_600 around 0.6 , the cultures were induced and checked for florescence.
Primers for two polarization tags (StpX and PflI) were created using "Genious" software and ordered.
Touch down was PCR done with primers listed above and wild type and pxyfpc-2 transformed CB15N and results were checked with gel electrophoresis.
Petri dishes have been filled with CB15N growth media PYE, pxyfpc-2 CB15N culture , and kanmycin. Glass slides were also placed in the petridishes in efforts to emulate monolayer production in paper below.
- citation needed*
Part 3
Results
pxyfpc-2 CB15N was plated on Kanamycin plate and grew many colonies. pvcfpc-2 CB15N has yet to grow on Gentamicin plates. Neither have yet to express fluorescence when induced.
PflI and StpX gels have yet to show bands of the correct size.
Monolayer experiments have shown growth but not in monolayer and have fluoresced without induction. It is speculated that premature fluorescence was caused by a leaky promoter.
