Part:BBa_K4212044

SDP1

L1

Introduction to this part

This part contains PlepA promoter, RBS2, gRNA CDS, and Spy terminator.

Our Experiment Design

In order for Cas9 to introduce a double stranded break in the DNA and hence unlock the nuclease activity of D15, a gRNA sequence was designed to target the origin of replication of the plasmid hosting the self digesting circuitry. The rationale behind this approach lies in the mode of action of D15. Indeed, this exonuclease hydrolyses phosphodiester bonds of nucleotide chains starting from their 3’ end. In order to increase the efficacy of our system, we hypothesized that a cut in the ori impair the functionality of the replicative region, hence limiting plasmid retention and duplication post-sporulation. As a matter of fact, even limited digestion of this regulatory region could still lead to the inability of replication of our system. This would still allow to reach appropriate protein expression levels while avoiding failure in digesting activity towards plasmid circuitry.

In its finalized design, a gRNA expression cassette lies in a STK 1A vector, featuring the strong promoter PlepA (STK029), strong RBS TM_RBS2 (STK046), its gRNA sequence and a Spy terminator (STK079).

A gRNA linear fragment was ordered from IDT with customized overhangs allowing entry into the Subtitoolkit. The fragment was resuspended in water to a concentration of 50 fmol/µL and used in a level 0 reaction. The assembly was then transformed and colonies screened via Phire PCR. The small size of the gRNA sequence made it hard to bands, run gel slower. ‍

Figure 1. The scheme of our gRNA assmebly design

Our Experiment Result

Positive transformants were inoculated in LB Cm and miniprepped before being sent for sequencing. Based on sequencing data, correct, unmutated samples were deposited in our glycerol stock collection. This indicated successful entry and domestication of a new part into the Subtitoolkit. Subsequently, vector L1A was assembled via a Golden Gate reaction to construct the transcriptional unit which would be expressed in the self digesting circuitry.

A molar ration of vector:insert of 1:2 was used. The reaction mix was then transformed in competent TOP10 cells and plated onto selective LB Amp media for recovery. Again, colonies were picked and screened via colony PCR harnessing Phire Master Mix (Thermofisher).

Positive hits were cultured overnight, mini-prepped and restriction digested to confirm whether the insert was successfully introduced in the L1A vector. (image)

Finally, samples that passed restriction digest screening were cultivated overnight for culture collection deposit.

Figure 2. The result of our gRNA assmebly design

References

[1] https://parts.igem.org/Part:BBa_K823002

[2] Homuth, G., Heinemann, M., Zuber, U. & Schumann, W. 1996 (n.d.) The genes lepA and hemN form a bicistronic operon in Bacillus subtilis. Microbiology. 142 (7), 1641–1649. doi:10.1099/13500872-142-7-1641.

[3] Qin, Y., Polacek, N., Vesper, O., Staub, E., Einfeldt, E., Wilson, D.N. & Nierhaus, K.H. (2006) The Highly Conserved LepA Is a Ribosomal Elongation Factor that Back-Translocates the Ribosome. Cell. 127 (4), 721–733. doi:10.1016/j.cell.2006.09.037.

[4] https://parts.igem.org/Part:BBa_K2680400

[5] Quijano, J.F. & Sahin, O. (2021) Genetically Intact Bioengineered Spores of Bacillus subtilis. ACS Synthetic Biology. 10 (4), 778–785. doi:10.1021/acssynbio.0c00578.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 163
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]