Part:BBa_K3286046:Design

mRFP-GFP divergent, Bacteriophage Lambda promoters, operators and 5'UTR's

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 7
  Illegal AgeI site found at 119
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1723
  

Design Notes

The native promoter regions, including the operator regions (OL and OR) and the 5'UTR's have been used in this construct. The start of either the mRFP or the GRP are at same position of as the native proteins downstream the PL and the PR promoters.

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:

• part:BBa_K3286040: F. novicida dCpf1 (dCas12a) protein
• part:BBa_K3286041: F. novicida Cpf1 (Cas12a) CRISPR array
• part:BBa_K3286046: mRFP-GFP divergent, Bacteriophage Lambda promoters, operators 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

Bacteriophage Lambda sequences were obtained from NCBI Reference Sequence: NC_001416.1 and synthetically synthesised in a gBlock. The mRFP and the GFP were obtained from iGEM supervisor.

References

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

Liu, X., Jiang, H., Gu, Z., & Roberts, J. W. (2013). High-resolution view of bacteriophage lambda gene expression by ribosome profiling. Proceedings of the National Academy of Sciences, 110(29), 11928–11933. https://doi.org/10.1073/pnas.1309739110

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