Difference between revisions of "Part:BBa K1463601:Design"

(→‎Design Notes)
(→‎Design Notes)
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To make the fliC biobrick, fliC was amplified by PCR using the proofreading Phusion polymerase with DS941 genomic DNA as template. The forward primer incorporated the prefix and added the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] ribosome binding site (RBS) and a scar sequence just upstream of fliC. The reverse primer incorporated the suffix and removed one undesirable Pst1 restriction site in fliC.
 
To make the fliC biobrick, fliC was amplified by PCR using the proofreading Phusion polymerase with DS941 genomic DNA as template. The forward primer incorporated the prefix and added the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] ribosome binding site (RBS) and a scar sequence just upstream of fliC. The reverse primer incorporated the suffix and removed one undesirable Pst1 restriction site in fliC.
  
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We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry [http://www.https://parts.igem.org/Part:BBa_K777109 K777109]. These two changes appear to be mutations in K777109 and thus our biobrick (BBa_K1463600) is an improved version of K777109.
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We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry [https://parts.igem.org/Part:BBa_K777109 K777109]. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation.
  
 
https://static.igem.org/mediawiki/2014/thumb/9/9e/GU_%28figure1%29_primer_for_fliC.png/800px-GU_%28figure1%29_primer_for_fliC.png
 
https://static.igem.org/mediawiki/2014/thumb/9/9e/GU_%28figure1%29_primer_for_fliC.png/800px-GU_%28figure1%29_primer_for_fliC.png

Revision as of 20:44, 17 October 2014

FliC with RBS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1242
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 319
    Illegal AgeI site found at 727
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To make the fliC biobrick, fliC was amplified by PCR using the proofreading Phusion polymerase with DS941 genomic DNA as template. The forward primer incorporated the prefix and added the BBa_B0034 ribosome binding site (RBS) and a scar sequence just upstream of fliC. The reverse primer incorporated the suffix and removed one undesirable Pst1 restriction site in fliC.

We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry K777109. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation.

800px-GU_%28figure1%29_primer_for_fliC.png

Source

E. coli DS941 genomic DNA

References