Team:AUC TURKEY/Measurement

37 °C

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.

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Medium Temperature IPTG Presence +/- Total Amount of Protein RFP Fluorometric Measurement RFP/Total

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4
14,09 22,32 1,584

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4
11,349 10,36 0,912

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25
17,87 14,35 0,802

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25
11,054 4,313 0,390

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37
16,808 40,2 2,391

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37
15,216 17,36 1,140

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41
7,637 44,83 5,870

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41
5,557 11,13 2,002

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50
5,463 60,06 10,993

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50
7,997 20,95 2,619





1. 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.

Characterization

We characterized the Toehold and Toehold+trigger genes of the ATOMS-TURKIYE team. 5 ml cultures containing the Toehold and toehold+trigger parts were incubated at 37 C for 16 hours. 1mM of iPTG was added at the end of the 16. hour. Another 4 hour incubation was conducted. The proteins in the incubated cultures were then isolated. A flurometric measurement was conducted at 473 nm of emission and 510 nm of excitation values. The difference in between the measurements for the protein isolates of the two parts can be seen below. Sample Name Total Protein Concentration GFP Flurometric Measurement GFP/TOTAL Toehold 7,1 0,2705 0,038099 Toehold trigger 3,5 9,031 2,5802

The difference in between the rates of the two samples is 1 to 68.