Team:Judd UK/Pages/Results

Results

First Culture of Colonies

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One of the first tests we performed on our plasmids was to produce colonies of E. coli which did not produce the chromoprotein. To do this, we cultured our bacteria on ampicillin agar that had a Fe2+ concentration of 0.1 M, however, we were unable to observe any growth compared to our control experiment. After further research, we discovered that lethal iron concentrations for the bacteria were just 1mM[1] so it is clear why this experiment failed. Another issue with this experiment was that the agar clumped together in the iron plates; this may be due to the fact that the iron may have provided nucleation sites for the agar, causing it to solidify unevenly.

Determining the identity of our constructs

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On the right is our attempt to transfer our constructs from the ampicillin resistant to a chloramphenicol-resistant plasmid; we generated several fragments during digestion of plasmids 1, 2 and 3 which when ligated with the chloramphenicol-resistant plasmid could have produced 2 different possible outcomes. In order to confirm the identity of our constructs, we extracted DNA from colonies using a miniprep kit. However, the identities of construct 2 and construct 3 were likely to be correct as they would appear blue in the absence of iron, (there is no Fe2+ to cause dimerization of the FUR protein and therefore no inhibition of amilCP synthesis in ligation 3 and there is no LacI protein present in our samples which would also inhibit the production of amilCP in ligation 2, therefore, colonies that had the correct plasmid would appear blue) and ran a gel electrophoresis on our sample. Using EcoR1 and Pst1 to generate our fragments we expected to generate (construct 1,2 and 3 were 1571, 1010 and 1090 base pairs respectively; the ampicillin resistant backbone is 2752 base pairs and the chloramphenicol resistant backbone is 2070 base pairs) and tests 1.1, 1.3, 2.1, 2.2 and 3.1 confirmed the identity of our constructs.

Testing our construct’s response to Iron

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We tried to replicate the Evry 2013 team’s experiment to test the responsiveness of our construct using our plasmids. We used the same volume of culture medium for each test (3ml of LB) with the corresponding concentration of as shown to the right.  We then picked a colony from our first culture of plasmid 3 and put one into each concentration of Fe2+. We then cultured each of them for 48 hours and then centrifuged the samples and removed the supernatant to concentrate the chromoprotein. Unfortunately, we were unable to discern any difference in our samples. This was likely because we were unable to control the number of cells per a sample, which would have altered the amount of chromoprotein produced per a sample. In addition, the time period over which this experiment was conducted may have been too long for any discernible difference to be identified, and there are limited nutrients within each sample which would lead to the eventual equal production of our chromoprotein.

In our next test we worked on these issues with our experiment and improved on our method; to control the cell number per sample we prepared our cell cultures in solution and used equal volumes of the cell culture per sample. In addition, we tested all of our recombined plasmids, and took samples at different times over a 24-hour period and therefore we should be able to see a difference in the rate of chromoprotein production. We also used a chelator called EDTA under the suggestion of Dr Jonathon McMaster from the University of Nottingham as he thought that the addition of a substance that binds iron to it may amplify the results of our test.

This test was to demonstrate the effects of co-transforming both our plasmids into both bacteria to compare the behaviours of our plasmids; we expected samples with plasmid 1 by itself to appear white like normal cultures of bacteria as it would produce LacI and no chromoprotein in response to iron. Plasmid 2 should have appeared the bluest as the production of amilCP is inhibited by the presence of LacI, which these bacteria could not synthesise; finally, plasmid 3 should be blue as well but to a lesser extent than samples with plasmid 2 as the production of amilCP is now linked to the concentration of Fe2+ ions.

After culturing and centrifuging our samples, we discovered that it was still difficult to discern any difference between the samples especially during the first sampling time (12 hours post-culture) as the quantity of protein produced was so small.

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This led us to prepare another set of samples without the presence of EDTA, but with an iron concentration of 10-6 M as the constructs appeared to respond best at this concentration. After culturing the bacteria for 22 hours and centrifuging them, the ampicillin culture appeared white, while the other two appeared blue, although the co-transformed bacteria were less blue due to the LacI produced by the first plasmid. We can, therefore, conclude that our co-transformed bacteria respond to iron when compared to the bacteria with the other 2 plasmids individually.

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Having learned from our mistakes in previous experiments, our final experiment to test the effects of Fe2+ included double the volume of cells used in previous experiments and no EDTA. We used plasmid 2 as our control as we knew that iron had no effect on the production of amilCP in this construct and we compared that to the co-transformed bacteria (plasmids 1+2). Again, we compared the quantity of amilCP produced by each of the samples.

Like previous experiments , samples taken at 15-21 hours (8:00-14:00) produced colonies that appeared white when centrifuged; this is due to the cells themselves and perhaps the LacI protein. Unfortunately, although some amilCP was produced by the samples at 24 hours and beyond, there was not enough present for the colour to register on the camera; when we compare these samples with bacteria that only had plasmid 2 and therefore exhibited no response to iron, producing only chromoprotein, it appears that our use of an inverter in our project added a significant delay between chromoprotein production and initial culture.

Interpretation

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References

<a name="_ftn1" title="">[1]</a> http://www.bioline.org.br/request?se08030

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