Part:BBa_K1806004:Experience

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Cloning

The composite system was ligated to the cloning vector psB1C3, to be transformated and then verified with Colony PCR. The verified ligation was then moved on to the digestion step to be cut with the EcoR1 and Pst1 restriction enzymes. The gel run of the cut parts were portrayed down below. The part was tagged as G-Block 6 and sample 6-2 yielded accurate results.

Functional Assay

The bacteria that were transformated with G-Block 7 were cultured in 5 ml and incubated for 13 hours. After this incubation, the cultures were incubated at respective temperatures for 4 hours and were given 50uM of iPTG twice with 4 hours periouds. There were a total 5 different temperatures that the cultures were incubated in: 4, 25, 37, 42 and 50 C. The proteins in the cultures were then isolated and the protein concentrations were measured. Data on RFP concentration were acquired at 584 nm of emission and 607 nm of excitation. The acquired values were divided to the total amount of protein to acquire the following ratio.

Tabled Data of the Protein Analysis

Medium Temperature	iPTG Presence +/-	Total Amount of Protein	RFP Fluorometric Measurement	RFP/Total
4	+	14.09	22.32	1.584
4	-	11.349	10.36	0.912
25	+	17.87	14.35	0.802
25	-	11.054	4,313	0,390
37	+	16.808	40.2	2.391
37	-	15.216	17.36	1.140
41	+	7.637	44.83	5.870
41	-	5.557	11.13	2.002
50	+	5.463	60.06	10.993
50	-	7.997	20.95	2.619

The graph shows the variance in density concentrations of RFP, caused as a result of the functioning of the iPTG Inducible Promoters in different temperatures. The functioning of the promoters increased cumulatively with increased temperatures.

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