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.

<img src="" alt="File:Judd uk iron testing.jpeg"><img src="" alt="File:Judd uk culture 2.jpeg">

<img src="" alt="File:Judd uk results summary final expt.jpeg">

Having learned from our mistakes in previous experiments, our final experiment to test the effects of Fe2+ included double the volu

me of cells used in previous experiments and no EDTA. We used plasmid 2 as our control as we kne

w 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.

In order to try and demonstrate that there was a difference in our samples, we also spread out our centrifuged pellets. It does appear that samples with the higher concentration of iron appear slightly darker, although colour can be subjective to the individual inspecting our results and the differences are very marginal. If we were able to quantify the chromoprotein using a plate reader, we may have been able to demonstrate definitively if there was any correlation between production of chromoprotein and the  concentration of iron.

It was difficult for us to exhibit any response to iron throughout our experimentation with our constructs; our first transformation using plasmid 3 which produces amilCP in the absence of iron produced normal looking colonies for at least two days before any visible amilCP was produced. Consecutive colonies picked from this initial culture yielded amilCP within a day, as demonstrated by our final experiment using the control sample. Our secondary PI has had previous experience using competent cells and suggested that this was normal behaviour for the cells.

When combined with the inverter sequence, there were large delays between initial culture and production of amilCP and below 48 hours of culturing, there was little expression of amilCP. The use of a double inverter in our project may have been responsible for the lack of consistency with our experiments with one experiment suggesting that amilCP would be produced by co-transformed cells (i.e. cells with the FUR repressed construct and LacI repressed construct) within 24 hours, although to a lesser extent than cells which lacked the first plasmid, while the other suggested that amilCP would not be visible until at least 24 hours later. This behaviour of our constructs would suggest that the repression of our constructs was not particularly good; this behaviour should be investigated further in the future to better characterise these parts.

An improvement on our construct would be perhaps to remove the second plasmid and try to fit our constructs on one plasmid using an iron responsive promoter region as opposed to a model which uses a double inverter to help remove this potential error.

Another improvement that could be made to our project, was that our range of iron concentrations did not accurately reflect those in patients whose saliva we would be analysing (between 1.8 mM to 63 mM for IDA and thalassemia major respectively); our experimentation method was based on work done by the Evry 2013 team using the same range of iron concentrations (10-10 M to 10-4 M) as we though that by using previous concentrations of iron that worked we would be able to observe clear differences in our samples. Evidently this was not the case; to improve on our methods, it would have been better to use a plate reader to standardise the number of cells per a test as well as quantify the production of amilCP.

Our in vivo tests showed little response to iron and the fact that our range of values that we would have to measure lies outside survivable bounds for our project suggests that our project would not be viable as a clinical test. However, the aim of our project was to produce a cell free system that would be able respond to iron; in spite of research that demonstrates that the rate of transcription is 10-100 times slower in vitro than in vivo, the use of a cell free system may be able to measure ranges of values that would normally be toxic for bacteria, however it is more likely that the delay shown by our constructs would have been amplified by the cell free test.