Difference between revisions of "Part:BBa K3286041:Design"
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Another system like our system was already present in the iGEM repository (BBa_K2003001), but this part contained BsaI sites and was not compatible with our backbone.
 
Another system like our system was already present in the iGEM repository (BBa_K2003001), but this part contained BsaI sites and was not compatible with our backbone.
  
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<h1> Inhibition of bacteriophage Lambda's regulatory region by Fn-dCpf1 (Cas12a) </h1>
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The following parts have been used and validated in our experiments to inhibit Red Fluorescent Protein (mRFP) and Green fluorescent protein (GFP) production downstream bacteriophage Lambda's promoters, operator regions (OL and OR) and 5'UTR's:
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<ul>
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<li>'''[[part:BBa_K3286040]]:''' F. novicida dCpf1 (dCas12a) protein
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<li>'''[[part:BBa_K3286041]]:''' F. novicida Cpf1 (Cas12a) CRISPR array
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<li>'''[[part:BBa_K3286046]]:''' mRFP-GFP divergent, Bacteriophage Lambda promoters, operators and 5'UTR's
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</ul>
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<ul><p>In addition, 5 different spacers were produced with part BBa_K3286041.
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New (d)Cpf1 (Cas12a) spacer constructs were produced by cloning of either annealed oligo’s or gBlock fragments into a BbsI digested BBa_K3286041 part.
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</p></ul>
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<ul><ul>The following Cpf1 spacer constructs were efficiently produced via this method and validated for their targeting:</ul>
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<ul><ul><ul><li>'''PL:''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT '''ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT</li>
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<li>'''PR:''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT '''TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT</li>
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<li>'''PL+PRM:'''  GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT '''ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT AAT CTA TCA CCG CAA GGG ATA AAT ATC TAA''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT </li>
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<li>'''PL+PR:'''  GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT '''ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT </li>
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<li>'''PRM+PR:''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT '''AAT CTA TCA CCG CAA GGG ATA AAT ATC TAA GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC''' GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT</li>
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</ul></ul></ul>
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<p>Cultures of <em>Escherichia coli</em> DH5a cells containing combinations of Part BBa_K3286040 with part BBa_K3286041 or of its derivatives (Cpf1 spacers PL, PR, PL+PRM, PL+PR and PRM+PR), resulted in significant differences in fluorescent protein production by part BBa_K3286046.
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Compared to controls without spacer part BBa_K3286041 or a derivate of it, were part BBa_K3286040 in combination with spacers PL, PL+PRM or PL+PR able to inhibit mFRP production by 98.3%, 96.7% and 95,3% respectively. In addition, GFP was significantly inhibited by spacers PR, PL+PR and PRM+PR by 96%, 95,4% and 96% respectively. </p>
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[[File:RFP-GFP-Lambda inhibition dCpf1.jpeg|750px|thumb|center|<b>Figure 1:</b> Inhibition of bacteriophage lambda promoters by Fn-dCpf1 (Cas12a). The promoters, operators (OL and OR) and 5'UTR of Bacteriophage lambda have been fused into a divergent RFP-GRP expressing plasmid and targeted by Fn-dCpf1 (Cas12a). Values are OD normalized and corrected for a control without a target spacer present. ]]
  
  

Revision as of 08:51, 17 October 2019


F. novicida Cpf1 (Cas12a) CRISPR array


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

Due to the fact that repeat regions are difficult to chemically synthesize, hard to clone and in general generate high number of errors, we were looking for a simpel and modular system for Cpf1 spacer-repeat production. Another system like our system was already present in the iGEM repository (BBa_K2003001), but this part contained BsaI sites and was not compatible with our backbone.

Inhibition of bacteriophage Lambda's regulatory region by Fn-dCpf1 (Cas12a)

The following parts have been used and validated in our experiments to inhibit Red Fluorescent Protein (mRFP) and Green fluorescent protein (GFP) production downstream bacteriophage Lambda's promoters, operator regions (OL and OR) and 5'UTR's:


    In addition, 5 different spacers were produced with part BBa_K3286041. New (d)Cpf1 (Cas12a) spacer constructs were produced by cloning of either annealed oligo’s or gBlock fragments into a BbsI digested BBa_K3286041 part.

      The following Cpf1 spacer constructs were efficiently produced via this method and validated for their targeting:
        • PL: GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT
        • PR: GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT
        • PL+PRM: GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT AAT CTA TCA CCG CAA GGG ATA AAT ATC TAA GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT
        • PL+PR: GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT ACA TAA ATA CCA CTG GCG GTG ATA CTG AGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT
        • PRM+PR: GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT AAT CTA TCA CCG CAA GGG ATA AAT ATC TAA GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT TCA CCG CCA GAG GTA AAA TAG TCA ACA CGC GTC TAA GAA CTT TAA ATA ATT TCT ACT GTT GTA GAT


    Cultures of Escherichia coli DH5a cells containing combinations of Part BBa_K3286040 with part BBa_K3286041 or of its derivatives (Cpf1 spacers PL, PR, PL+PRM, PL+PR and PRM+PR), resulted in significant differences in fluorescent protein production by part BBa_K3286046. Compared to controls without spacer part BBa_K3286041 or a derivate of it, were part BBa_K3286040 in combination with spacers PL, PL+PRM or PL+PR able to inhibit mFRP production by 98.3%, 96.7% and 95,3% respectively. In addition, GFP was significantly inhibited by spacers PR, PL+PR and PRM+PR by 96%, 95,4% and 96% respectively.


    Figure 1: Inhibition of bacteriophage lambda promoters by Fn-dCpf1 (Cas12a). The promoters, operators (OL and OR) and 5'UTR of Bacteriophage lambda have been fused into a divergent RFP-GRP expressing plasmid and targeted by Fn-dCpf1 (Cas12a). Values are OD normalized and corrected for a control without a target spacer present.


    Source

    Chemically synthesized F. novicida CRISPR array

    References

    Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., … Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell, 163(3), 759–771. https://doi.org/10.1016/j.cell.2015.09.038