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Composite

Part:BBa_K1806004

Designed by: İBRAHİM YASİR ORHAN   Group: iGEM15_AUC_TURKEY   (2015-09-17)
Revision as of 20:49, 20 September 2015 by Abdulkadir (Talk | contribs) (Cloning)

T7+ ibPB RNA Thermometer + pelB + MCS + 6xHis

The part is to act as a backbone for the T7+ibPB RNA Thermometer+pelB+6xHis system. The MCS(Multiple Cloning Site) as the ligation region for the parts to be integrated into the system. The part that is added to the backbone will be translated in the presence of higher temperatures. The pelB signal peptide is present for the produced protein to be carried out of the cell. The 6xHis is for protein analysis.

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.

AUC TURKEY 6 colony.png 6004-2.png

AUC TURKEY 6,1,3 cutcheck.png 6004-4.png

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

6005 FA1.jpg

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.

20150910 104908ddxdfghc.jpg


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2
    Illegal BamHI site found at 252
    Illegal XhoI site found at 277
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 219
  • 1000
    COMPATIBLE WITH RFC[1000]


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