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Research to produce the protein that synthesize Lipoic Acid.

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Introduction

RESEARCH TO PRODUCE THE PROTEIN THAT SYNTHESIZE LIPOIC ACID. REPORT INTRODUCTION My principle objective was to isolate the DNA encoding Lipoic Acid Synthase (lipA) in Aeropyrum pernix (a thermophilic bacterium), and to over express the protein (LipA). Lipoic acid is a vitamin required for the healthy growth of prokaryotic and eukaryotic cells. It is synthesised by LipA in the following reaction: DNA from Aeropyrum pernix was kindly provided by Dr. M. Kreik (Southampton University), which contained the required gene. The initial practical steps for the project were: 1). Amplifying lipA gene by polymerase chain reaction (pcr). 2). Separating the pcr products on an agarose gel. 3). Isolating the required gene present in the band. Subsequently, I carried out following steps 4). Capturing the gene in a TOPO plasmid. 5). Inserting the plasmid into Escherichia coli. 6). Allowing it to grow in a medium supplemented with ampicillin These steps allowed us to isolate bacterial colonies having lipA inserted into the TOPO plasmid. I then purified the plasmid from the rest of the cell components (Mini-Prep Kit). The next steps were required to transfer the LipA gene from the TOPO plasmid (which is good for cloning) to the pBAD-His plasmid, which is good for protein production. By using endonucleases, (restriction enzymes) the plasmids were digested I then separated the gene from the rest of the plasmids using agarose gel electrophoresis (as used above). ...read more.

Middle

Smaller fragments move quickly through the gel, but the larger fragments are held back by the gel and move slowly. Markers of standard sizes are used to provide a size scale on the gel. The different bands are stained using ethidium bromide and visualised on the gel using ultra violet light. The gene we were looking for was 1000 bases long. In order to get the required gene we separated our products that we got from pcr on the agarose gel. We were expecting to get something near 1000 bases, as our gene was 1000 bases long. We did several experiments to get a fragment at 1000 point. Pfu and vent never worked but taq worked well. We changed conditions, primers and dna concentrations, annealing temperatures but we didn't got any results so we decided to use taq. The gel on which the products produced by pcr using taq was run is shown below Gel 1. Separation of PCR products using Taq polymerase. Lane 1, size markers, lane 2, blank, lanes 3 and 4: PCR products. At this point we had the bands that was required so i ran the products again on a low melting point agarose gel, to cut out the required bands. We then ligated the lipA gene into the topo plasmid which was already opened to accept it. ...read more.

Conclusion

Then i took the PBAD-HIS plasmid that already had the ampicillin gene cut it open using restriction endonuclease and ligated the ISC gene and lipA gene as well into this plasmid. At this point the PBAB-HIS plasmid had the lipA ,isc and the ampicillin resistance genes. After this we transferred this plasmid inside e-coli bacteria and allowed it to grow in a medium containing ampicillin so that only the colonies having the lipA gene grew. Then I separated these colonies containing our plasmid and allowed them to grow separately and to produce our LipA protein. After this I started to purify the protein. Bacterial cells do not produce only one type of protein but produce many different proteins as well. The LipA produced has a His-tag on it which binds with nickel very strongly. I then passed all the proteins that were produced through the resin containing nickel in it. Only the LipA protein binds to the resin, others should be washed off. There may be some other proteins which have bound to the nickel resin, the resin was washed with 20mM and 50mM imidazole to remove non His-tagged proteins. The LipA was eluted with 500mM imidazole which was strong enough to remove our protein. This protein was separated on a protein gel, the diagram of this gel is shown below the length of our protein is 32000 base pairs in size. LipA Gel 4 LipA separated on a protein gel ...read more.

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