Difference between revisions of "Part:BBa K215002"
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<partinfo>BBa_K215002 short</partinfo>
 
<partinfo>BBa_K215002 short</partinfo>
  
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For example, assume our gene (E0040) starts with:
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Any favorite protein (afp) can be inserted into the NheI (compatible with XbaI and SpeI, but make sure the resulting product stays in frame) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP). The series of tags are a Nano tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: BB#). To use in E. coli we recommend using the composite part BB#, which already has a strong promoter and RBS attached. In order for the secretion tag to function this part needs to be used in conjunction with BB#.  
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5'-atgcgtaaaggagaagaacttt...-3'
+
  
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The design process would be:
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Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct will result in a stop codon at the resulting scar site.  Therefore, to insert a gene a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:
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# Start with the XbaI site: 5'-TCTAGA-3'
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# Add ~20bp of the gene immediately after the XbaI site, starting _after_ the start codon (atg), and try to end on a G or C: 5'-TCTAGAcgtaaaggagaagaacttt-3' for E0040 from above.  These ~20bp will determine the melting temperature for the primer.
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# Add 6-8 random nucleotides at the start.  Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
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# Tweak the number of nucleotides until the melting point roughly matches that of Vr.
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Then to add the gene to the construct, again assuming the gene is a biobrick with standard suffix:
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#Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon.
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# PCR with the designed forward primer and Vr
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##Forward Primer:  <8 random bp's> - <NheI/XbaI/SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
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# Then run the PCR product in a digest with XbaI and PstI, and digest this part (the display construct) with NheI and PstI. The XbaI site has a sticky end that binds with NheI.
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##Reverse Primer:  <15-21bp complementing your gene of interest, ending BEFORE the stop codon (make sure it is a muliple of three to stay in frame)> - <NheI/XbaI/SpeI> - <8 random bp's>
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# Standard ligation and transformation.
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#Amplify the gene of interest
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#Clone into the NheI site of this construct.
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#Screen colonies with either the Forward Primer+VR or the VF2+Reverse primer for inserts in the appropriate direction.
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#Your fusion protein is tagged and ready for secretion and streptavidin binding!
  
  

Revision as of 05:34, 1 October 2009

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

Any favorite protein (afp) can be inserted into the NheI (compatible with XbaI and SpeI, but make sure the resulting product stays in frame) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP). The series of tags are a Nano tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: BB#). To use in E. coli we recommend using the composite part BB#, which already has a strong promoter and RBS attached. In order for the secretion tag to function this part needs to be used in conjunction with BB#.

Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct will result in a stop codon at the resulting scar site. Therefore, to insert a gene a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:

  1. Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon.
    1. Forward Primer: <8 random bp's> - <NheI/XbaI/SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
    2. Reverse Primer: <15-21bp complementing your gene of interest, ending BEFORE the stop codon (make sure it is a muliple of three to stay in frame)> - <NheI/XbaI/SpeI> - <8 random bp's>
  1. Amplify the gene of interest
  2. Clone into the NheI site of this construct.
  3. Screen colonies with either the Forward Primer+VR or the VF2+Reverse primer for inserts in the appropriate direction.
  4. Your fusion protein is tagged and ready for secretion and streptavidin binding!


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 196
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]