Difference between revisions of "Part:BBa K3286040:Design"
 
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The DNA sequence of Fn-dCpf1 (Cas12a) was obtained from (Zetsche et al., 2015).
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__NOTOC__
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<partinfo>BBa_K3286040 short</partinfo>
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<partinfo>BBa_K3286040 SequenceAndFeatures</partinfo>
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<h1> Inhibition of bacteriophage Lambda's regulatory region by Fn-dCpf1 (Cas12a) </h1>
 
<h1> Inhibition of bacteriophage Lambda's regulatory region by Fn-dCpf1 (Cas12a) </h1>
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<ul><ul>The following Cpf1 spacer constructs were efficiently produced via this method and validated for their targeting:</ul>
 
<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
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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
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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  
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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  
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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
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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>
 
</ul></ul></ul>
 
</ul></ul></ul>
  
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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. ]]
 
[[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. ]]
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===Source===
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The DNA sequence of Fn-dCpf1 (Cas12a) was obtained from (Zetsche et al., 2015).
 +
 +
 +
===References===
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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
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Leenay, R. T., Maksimchuk, K. R., Slotkowski, R. A., Agrawal, R. N., Gomaa, A. A., Briner, A. E., … Beisel, C. L. (2016). Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Molecular Cell, 62(1), 137–147. https://doi.org/10.1016/j.molcel.2016.02.031

Latest revision as of 08:54, 17 October 2019


F. novicida dCpf1 (dCas12a) protein


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 16
    Illegal PstI site found at 280
    Illegal PstI site found at 1543
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 16
    Illegal NheI site found at 1549
    Illegal PstI site found at 280
    Illegal PstI site found at 1543
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 16
    Illegal BglII site found at 1502
    Illegal BglII site found at 1536
    Illegal BglII site found at 1622
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 16
    Illegal PstI site found at 280
    Illegal PstI site found at 1543
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 16
    Illegal PstI site found at 280
    Illegal PstI site found at 1543
    Illegal NgoMIV site found at 3231
  • 1000
    COMPATIBLE WITH RFC[1000]


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

    The DNA sequence of Fn-dCpf1 (Cas12a) was obtained from (Zetsche et al., 2015).


    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

    Leenay, R. T., Maksimchuk, K. R., Slotkowski, R. A., Agrawal, R. N., Gomaa, A. A., Briner, A. E., … Beisel, C. L. (2016). Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Molecular Cell, 62(1), 137–147. https://doi.org/10.1016/j.molcel.2016.02.031