Difference between revisions of "Part:BBa K2996700"

 
 
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<partinfo>BBa_K2996700 short</partinfo>
 
<partinfo>BBa_K2996700 short</partinfo>
  
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Flexible Linker 4 (FL4) has been proved to be effectively separate bifunction proteins. This is a long linker (25 aa), which better accommodates RpoA:promoter interaction. Amino acid sequence of FL4 is:LSGGGGSGGGGSGGGGSGGGGSGGGGSAAA.
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FL4 contains 5 repeats of GGGGS, which is a common design of flexible protein structures.
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It links RpoA and dCas9.  
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Unfortunately, highly-repeating sequence is difficult to deal with during molecular cloning. We have tried these two DNA sequences for elongated FL4 (eFL4), however overlap extension PCR during these two trials both end up in failure.
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<br> >eFL4
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CTGAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGCGGCGGCG
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<br> >eFL4 Optimized by GeneScript
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TTATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGG TGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGCGGTGGTGGTGGTTCTGGTGGTG GTGGTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCAGGTGGTGGTGGTTCGGGTGGT GGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGGTGGTTCGGGTGGTGGTGGTTCTGGTGG TGGTGGTTCAGCAGCAGCA
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So, we optimized FL4 sequence ourselves, with a simple Python program that randomly select codons for FL4:
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CTGTCCGGAGGAGGCGGGTCCGGCGGGGGTGGCTCCGGAGGCGGGGGATCGGGTGGAGGGGGTTCTGCTGCCGCG
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===Results===
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<center>{{#tag:html|<img style="max-width: 50%" src="https://static.igem.org/mediawiki/parts/a/ae/T--SJTU-BioX-Shanghai--result_gel1.png" alt="" />}}</center>
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<center><b>Figure 1.</b> <i> Overlap Extension PCR for rpoA-FL4-dCas9</i> </center>
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<p> Based on this, we constructed plasmid pActivator without sgRNA and pActivator with sgRNA.
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===Usage===
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Using overlap PCR, RpoA is linked to dcas9 through a modified flexible Linker 4 (FL4), which has been proved to be effectively separate bifunction proteins.  
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Now we got the transcription activator BBa_K2996701.
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===Usage and Biology===
 
  
 
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===Functional Parameters===
 
 
<partinfo>BBa_K2996700 parameters</partinfo>
 
<partinfo>BBa_K2996700 parameters</partinfo>
 
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Latest revision as of 23:51, 21 October 2019


Flexible Linker 4 that separates RpoA and dCas9

FL4 contains 5 repeats of GGGGS, which is a common design of flexible protein structures. Unfortunately, highly-repeating sequence is difficult to deal with during molecular cloning. We have tried these two DNA sequences for elongated FL4 (eFL4), however overlap extension PCR during these two trials both end up in failure.
>eFL4 CTGAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGCGGCGGCG
>eFL4 Optimized by GeneScript TTATCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGG TGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTAGCGGTGGTGGTGGTTCTGGTGGTG GTGGTAGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCAGGTGGTGGTGGTTCGGGTGGT GGTGGTTCTGGTGGTGGTGGTAGTGGTGGTGGTGGTTCGGGTGGTGGTGGTTCTGGTGG TGGTGGTTCAGCAGCAGCA

So, we optimized FL4 sequence ourselves, with a simple Python program that randomly select codons for FL4: CTGTCCGGAGGAGGCGGGTCCGGCGGGGGTGGCTCCGGAGGCGGGGGATCGGGTGGAGGGGGTTCTGCTGCCGCG

Results

Figure 1. Overlap Extension PCR for rpoA-FL4-dCas9

Based on this, we constructed plasmid pActivator without sgRNA and pActivator with sgRNA.

Usage

Using overlap PCR, RpoA is linked to dcas9 through a modified flexible Linker 4 (FL4), which has been proved to be effectively separate bifunction proteins. Now we got the transcription activator BBa_K2996701.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]