Following the harvesting and re-suspending of the induced cultures the
bacteria were purified by running the sample through Nickel charged
resin. Both the wash buffer and the elution buffer had constant pH of 8.
This fits in the pH range in which GFP is stable in being from 6 to 10.
(Patterson et al. 1997)
After purification and elution of the GFP containing solution elute was
a colorless see-through liquid. It therefore failed to show a presence
of GFP that gives elute a bright green color. Furthermore, the SDS gel
failed to show a presence of a clear rGFP in the required, 27 kb elution
band. There is a faint band that can be noticed, however it could be
just an assembly of unrelated proteins or just not sufficient amount of
GFP to consider the expression a success.
Discussion: The aim of the experiment was not only to successfully
purify the rGFP but also to appreciate its unique properties and to gain
the understanding of common techniques used in modern cloning and
expression of recombinant proteins, which was successful as it
definitely provided an insight into common methods used in biochemical
laboratories.
Obtained results showed a partially successful experiment. The plasmid
transformation was successful and sufficient bacterial growth on the
antibiotic enriched media was observed as shown by Figure 3A. However
the expression and purification of the protein did not go according to
initial assumptions. There are multiple possibilities, why the
experiment did not go as planned.
While looking at the SDS-PAGE gel, Figure 3B one can notice that the
bands in “pre “and “post” induction samples are exactly the same. As
mentioned in the “results “section, low amounts of GFP are expressed
even without induction due to promoter polymerase overlapping. Similar
band widths in “pre and post” induction wells imply that the
inoculation did not happen correct and that possibly the amount of IPTG
was not sufficient to induce cells. Moreover the induction took place at
inappropriate OD and therefore had to be incubated at
370C overnight. This could result in the ampicillin
being used up. E.Coli, being incredibly responsive to its environmental
changes ejects the plasmid when theantibiotic is not present in its
environment. Potentially the prolonged incubation resulted in the
antibiotic being used up and the plasmid being ejected and hence no rGFP
production was observed. No production theory is further supported by
lack of visibly green, rGFP in the re-suspended pellets. Colleagues who
managed to obtain rGFP had a clear, bright green suspension. Suspension
obtained by myself was brownish yellowish. At this point it was still
unclear whether the experiment was successful since the color only did
not forejudge the outcome. Despite lack of visibly green solution, there
was a good probability that some amounts of rGFP were produced and will
become visible after the purification and will be observed after the
SDS-page analysis. The IMAC resulted in completely see-through, clear,
colorless elute which further indicated that the experiment has failed.
Obtained elute should have been a clear, bright green solution due to
the rGFP natural properties, and it was observed as such among
colleagues whose experiments were successful. Arguably there is a faint
bad present on the SDS-PAGE, however I believe that this is nothing more
than contaminants. Since the nickel resin is charged, the band is more
than likely to contain various other proteins that are result of a
non-specific binding. To examine the purity of a band further and to
fully determine whether the obtained band was a purified rGFP it is
essential to perform second chromatographic assay, such as size
excursion chromatography. At a current time it is impossible to fully
determine the nature of obtained band, it could indeed be a low amount
of rGFP but most likely it is a contaminant or a degradation product.
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