pGLO Transformation and Purification
Over three weekends, I attended workshops where I carried out two experiments. The first was a transformation of E.coli with the plasmid pGLO, which contains the gene GFP as well as another gene (beta lactamase) which codes for resistance to ampicillin, an antibiotic. GFP stands for ‘Green Fluorescent Protein’ and is found in a particular species of jellyfish, which are able to glow in the dark as a result of the GFP gene.
In the very beginning of the experiment, gel electrophoresis was carried out to confirm that there were pGLO plasmids in the sample given, to make sure that the experiment would yield results. The plasmids where dyed with brilliant Blue (FCF) to make them visible. After the electrophoresis had been running for half an hour, the gel was placed under UV light. The results show that the plasmids are in the sample, as they travelled along the gel. This is shown in the picture on the left.
To actually transform the E. coli bacteria with the GFP gene, calcium chloride was added to the plasmids. Adding it allows for the bacteria to take up the pGLO plasmid easier as it neutralises the negative charges on phospholipids on the bacterial cell wall. This increases permeability of the prokaryotic cell wall which allows the plasmid to enter the bacterium more efficiently. The pGLO plasmid is added to one of the test tubes, so is named ‘+pGLO’. However there is no pGLO plasmid added to the other test tube so is named ‘-pGLO’. This is so I had a control in my experiment and could observe a clear difference in the phenotype of the transformed bacteria after they had taken up the pGLO plasmid.
To help the bacteria take up the plasmid I moved them from the ice to a water bath which was at 42°C. This change in temperature results in heat shock, so it increases the permeability of the bacteria membrane so that it allows for the plasmid pGLO to be taken up easier.
The four test tubes (two are +pGLO and two are -pGLO) are added to 4 petri dishes with different compositions of agar in them. All of them have a nutrient base composite named LB so that they can grow. However, they differ from one another as one contains just LB (LB), another contains LB, ampicillin and arabinose (LBAA), and the remaining two just contain LB and ampicillin (LBA). The 4 petri dishes were left for enough time for colonies to grow on the agar and then were placed under UV light to observe the changes as shown in the picture below.
Using the results obtained from the experiment, one can see which bacteria expresses the GFP gene when the bacteria is put under UV light. Since no pGLO plasmid was added to the test tubes called ‘-pGLO’, the bacteria grown on that plate will not express the gene since GFP was present in the plasmid at all. So, both plates with the –pGLO bacteria will not express the gene anyway, which is expected. In the +pGLO tubes, the pGLO is taken up by the E.coli however bacteria on only one of the two plates actually expressed the GFP gene. In the LBAA plate, the gene was expressed, but in the LBA plate, it is not. This is because of the growth medium as LBAA contains arabinose but LBA does not. Arabinose is a sugar that activates the GFP gene, so without it the glowing phenotype cannot be seen. Further evidence to show that the pGLO plasmid has been taken up is the fact that bacteria has successfully grown on the +pGLO LBA plate but failed to grow on the –pGLO LBA plate. This is because as well as containing the GFP gene, the pGLO plasmid also contains beta lactamase, which allows for resistance to ampicillin (an antibiotic). Without the pGLO plasmid, the bacteria on the –pGLO plate was killed by the ampicillin on the plate due to lack of resistance. On the +pGLO plate, the plasmid is taken up and therefore the bacteria has grown. However it has not expressed the GFP gene because arabinose is required to activate it.
Gel electrophoresis was again carried out to confirm that GFP was present in the E.coli.This was done by taking colonies of bacteria from the plate where the GFP was expressed and buffer was added to it. It was then added to the gel where it was left for a week. When the gels were removed and viewed, a large band was observed at around 25kDa mark. This was the GFP protein as research shows that its molecular weight is about 27kDa.
For the second experiment, the GFP gene was removed from the bacteria and then purified to try to reach a state as close as can be to the original GFP that was transformed into the bacteria. After centrifuging twice and using hydrophobic interaction chromatography the three collection tubes at the end of the experiment had UV light shone on them.Collection tubes 1 and 2 showed a different result to that of three at the end of the experiment, the reason for this lies in the theory of the HCI matrix and the effect of the buffers that were washed through during hydrophobic interaction chromatography.
In conclusion, from these experiments carried out at the workshops, E.coli was successfully transformed with the plasmid pGLO, enabling the bacteria to be resistant to ampicillin and also to fluoresce under UV light (due to the presence of the GFP gene). Also, GFP was then successfully extracted from the same bacteria purified, giving an insight into some other modern day processes like the production of insulin.
Sophie Waring 12R1