Not Released
Sample In stock
Experience: Works
Not Used
Get This Part
Composite

Part:BBa_K1806005:Experience

Designed by: İBRAHİM YASİR ORHAN   Group: iGEM15_AUC_TURKEY   (2015-09-17)
Revision as of 18:14, 22 September 2015 by Skywalker (Talk | contribs) (→‎Cloning)


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1806005

Cloning

The already linear part was ligated to the pSB1C3 vector, and then transformated to be verified with Colony PCR. The RFP in the iGEM Registry contained Hind3, unlike the RFP in this part. For this reason, the part was cut with the EcoR1 and Hind3 restriction enzymes. The gel runs of the part were examined. The part was tagged as G-Block 7 and sample 7-6 yielded the accurate base pair length.

6005-1.png 6105-2.png

AUC TURKEY 7,6 cutcheck.png 6005-4.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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 720
    Illegal AgeI site found at 832
  • 1000
    COMPATIBLE WITH RFC[1000]


User Reviews

UNIQe412939cde854ebb-partinfo-00000001-QINU UNIQe412939cde854ebb-partinfo-00000002-QINU