Device

Part:BBa_K174007:Design

Designed by: The Newcastle 2009 iGEM team   Group: iGEM09_Newcastle   (2009-10-10)
Revision as of 19:39, 10 October 2009 by Goksel (Talk | contribs)

Degradation controller with integration site


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To test the integration, pmutin4 integration vector is selected. However after the integration pspac is left on the right side, free to drive the expression of downstream genes. To remove the effect of pspac promoter of pmutin4 vector, the device with ligation of sac integration site, araR repressible promoter and sspB CDS was amplified with PacI from one end. Hence by cutting pmutin4 with PacI, pspac promoter can be removed. For the other end, sacII was selected since it is compatible with PacI. PacI is just on the left side of Terminator0 on pmutin4, it will also be removed leaving Terminator 1 and Terminator 3 on the vector.

Sac, araR promoter+RBS, and sspB biobricks are combined as a device into the Biobrick compatible vector pSBa1AT3. The sequence of the device (without the biobrick prefix and suffix) is then amplified by PCR with dangling end primers with PacI at 5’ and SacII at 3’. Pmutin4 is cut with PacI and SacII and the backbone of pmutin4 without terminator_0 and pspac is ligated with the PCR product.

Newcastle 2009 integration 2.png


After the controller is placed into pmutin4, the vector will lose terminator0 and pspac and have the controller instead.

Newcastle 2009 integration 3.png


After the integration, the whole vector will be integrated onto the sac region on the chromosome as below.


Newcastle 2009 integration 1.png


Virtual construct of the device and pmutin4 integration vector.


Source

Ligated manually

References