Difference between revisions of "Part:BBa K1962015"
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We cloned the Ia-Immunity protein (<partinfo>BBa_K1962001</partinfo>) downstream of a pH sensitive promoter Pasr (<partinfo>BBa_K1231000</partinfo>) and a bile salt sensitive promoter PacrRA (<partinfo>BBa_K1231001</partinfo>) to generate the composite parts (<partinfo>BBa_K1962015</partinfo>) and (<partinfo>BBa_K1962011</partinfo>), respectively.
 
We cloned the Ia-Immunity protein (<partinfo>BBa_K1962001</partinfo>) downstream of a pH sensitive promoter Pasr (<partinfo>BBa_K1231000</partinfo>) and a bile salt sensitive promoter PacrRA (<partinfo>BBa_K1231001</partinfo>) to generate the composite parts (<partinfo>BBa_K1962015</partinfo>) and (<partinfo>BBa_K1962011</partinfo>), respectively.
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The immunity proteins for colicin Ia and E3 were cloned in front of the asr pH sensitive promoter. The immunity proteins were amplified with a HA tag fused onto the C-terminal of the immunity proteins to allow for visualisation of expression via western blotting. As shown in Fig 1 we were unable to detect expression of iA-Immunity protein.
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https://static.igem.org/mediawiki/2016/1/1d/File-T--Dundee--Results16.5.png
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Figure 1. Western blot of colicin Ia and E3 immunities with pasr: The col Ia and E3 immunities were amplified with a HA tag to detect expression via a western blot. The asr promoter was blotted alongside as a control. No expression of the immunities in front of the asr promoter was detected.
  
 
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Revision as of 21:54, 22 October 2016


A device for expression of Im-Ia in response to pH

This is a composite part for the production of the Immunity Protein to Colicin Ia in response to environmental pH changes. The composite part consists of a pH sensitive promoter (asr from biobrick BBa_K1231000, an RBS (BBa_B0030) and the Immunity Protein required to neutralise the bacteriocin activity of Colicin Ia (BBa_K1962001).


Usage and Biology

We cloned the Ia-Immunity protein (BBa_K1962001) downstream of a pH sensitive promoter Pasr (BBa_K1231000) and a bile salt sensitive promoter PacrRA (BBa_K1231001) to generate the composite parts (BBa_K1962015) and (BBa_K1962011), respectively.

The immunity proteins for colicin Ia and E3 were cloned in front of the asr pH sensitive promoter. The immunity proteins were amplified with a HA tag fused onto the C-terminal of the immunity proteins to allow for visualisation of expression via western blotting. As shown in Fig 1 we were unable to detect expression of iA-Immunity protein.

File-T--Dundee--Results16.5.png

Figure 1. Western blot of colicin Ia and E3 immunities with pasr: The col Ia and E3 immunities were amplified with a HA tag to detect expression via a western blot. The asr promoter was blotted alongside as a control. No expression of the immunities in front of the asr promoter was detected.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 215
    Illegal AgeI site found at 422
  • 1000
    COMPATIBLE WITH RFC[1000]