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). At this point it was inserted in a pBAD-His plasmid which had some other helpful genes already inserted into it. The plasmid was again used to transform E. coli and the bacteria were allowed to grow in a medium having ampicillin. Finally, the bacteria containing the correct plasmid were allowed to grow in bacterial culture medium and the protein was isolated using Nickel affinity chromatography.
The purified DNA of the thermophilic bacteria Aeropyrum pernix was provided. The lipA gene was amplified from this DNA using polymerase chain reaction.
1. Polymerase chain reaction
This is a technique to amplify the required gene. In this technique Stock solution containing the DNA Primers buffer and water is heated to a certain temperature so that the DNA double helix separate into two single strands of DNA. It is then cooled so that the primers can anneal to the single stranded genomic DNA so as the nucleotides to make two new DNA. The DNA polymerase then extends the primer to make a complete DNA helix. This process is repeated several times so that i ended up with amplified double stranded DNA, making enough of the gene for the next step.
An enzyme DNA Polymerase is used in this process to extend the primers to a complete DNA strand. I used three different enzymes in my research which are as follows.
1) pfu turbo DNA polymerase. (Not very powerful but precise and accurate)
2) taq DNA polymerase. (Very powerful but sometimes makes sequence mistakes)
3) Vent DNA polymerase. (Moderate but accurate)
We carried out many experiments in order to get the required lipA using three of these enzymes in several different experiments.
After getting the products from polymerase chain reaction we carried out a process called gel electrophoresis which shows us that we have our required gene or not
This is a process of separating the smaller fragments from the larger ones. The sample whose composition we wants to know is run on the gel. The fragments are loaded into a gel across which is an electrical potential. 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. The TOPO plasmid had the ampicillin resistance gene which was important for the next stage. The two ends of the PCR product were complementary to the two ends of the TOPO plasmid that joins together (ligates) at this stage, to give the recombinant plasmid. This plasmid had the lipA gene and the ampicillin resistant gene, it was transferred into E. coli bacteria and they were allowed to grow in a medium containing ampicillin, the colonies that survived was the ones carrying lipa gene. Some of the colonies had the recombinant plasmids, but others didn’t so only these colonies having lipA gene grew in a medium containing ampicillin .
Than we extracted the plasmids from the bacterial cell by the process of digestion. Following steps were carried out after this
1) cutting the plasmids using restriction enzymes
2) getting the products and running them on the agarose gel
The picture of the gel that we got by this process is shown below:
Gel 2. Restriction digestion of potential lipA:pBAD-TOPO plasmids. Samples 3 and 9 contain the 1000 bp fragment I required.
I then separated the ISC genes using the same technique as i used to isolate the lipA gene and ligated it into a pBAD-His plasmid.
As it is known that all the LipA proteins that are naturally made have got the iron sulphur cluster on them but the protein we are making is produces at artificially high levels, so we have to put the iron sulphur cluster on it. For this we have to insert the ISC genes into the plasmid. This isc gene is extracted from the e-coli DNA using the same method as we used for extracting the lipa gene. Once again pfu turbo dna polymerase and vent didn’t worked but this taq worked so we decided to use the product that we got by using taq. The picture of the gel on which these products were run is shown below.
Lane 1 Size 2 3 4 5 6 7 8
on this gel we can clearly see the bands containing the ISC gene.
I then ran the products on a low melting point agarose gel and separated the desired bands. 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.