Difference between revisions of "Part:BBa K174007:Design"

 
 
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===Design Notes===
 
===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.
 
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.
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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.
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Forward primer used: GGTGCTG-TTAATTAA-TTCTCATTTCTGCGCATGGC (Clamp sequence -  PacI restriction site - first 20 base from the Biobrick)
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Reverse primer used: TGTGAT-CCGCGG-TTACTTCACAACGCGTAATGCC (Clamp sequence - SacII restriction - last 22 base from the Biobrick)
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[[Image:Newcastle_2009_integration_2.png|300px]]
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After the controller is placed into pmutin4, the vector will lose terminator0 and pspac and have the controller instead.
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[[Image:Newcastle_2009_integration_3.png|500px]]
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After the integration, the whole vector will be integrated onto the sac region on the chromosome as below.
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Addition of arabinose to the medium  should activate AraR promoter region on the Biobrick and drive the expression of both sspB and lacZ. Expression of lacZ can be visually tested with blue coloured colonies.
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[[Image:Newcastle_2009_integration_1.png|400px]]
  
  

Latest revision as of 19:44, 10 October 2009

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.

Forward primer used: GGTGCTG-TTAATTAA-TTCTCATTTCTGCGCATGGC (Clamp sequence - PacI restriction site - first 20 base from the Biobrick)

Reverse primer used: TGTGAT-CCGCGG-TTACTTCACAACGCGTAATGCC (Clamp sequence - SacII restriction - last 22 base from the Biobrick)


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. Addition of arabinose to the medium should activate AraR promoter region on the Biobrick and drive the expression of both sspB and lacZ. Expression of lacZ can be visually tested with blue coloured colonies.


Newcastle 2009 integration 1.png


Source

Ligated manually

References