Difference between revisions of "Part:BBa K4212048"

Revision as of 10:21, 13 October 2022

SDP1

L2

Introduction to this part

This part contains SDP1, SDP2, SDP3, and SDP4.

Our Experiment Design

One key aspect of Sporadicate’s biocontainment approach is a self digesting circuit. This consists of a CRISPR/Cas9 system to perform a double stranded cut at the origin of replication of the plasmid used to express transcriptional units in the cell, as well as the exonuclease D15 which can then digest the plasmid DNA upon detection of a free 3’ end. By utilizing promoters specific to a late stage of sporulation (T3 - wherein forespore and mother cell are completely separated) and the forespore region, this system allows us to transiently produce proteins before the cell forms a spore. Once the sporulation process begins, the proteins belonging in the coat, already produced, can integrate accordingly and continue to be expressed in the mother cell, whilst any plasmid DNA found in the forespore will express the gRNA, Cas9 and D15 key to the self-digesting system. The final product is a spore that does not contain any foreign DNA but that does display heterologous proteins on its surface. There are thus 4 transcriptional units integral to the self-digesting plasmid: gRNA, D15, Cas9 and protein of interest.

Our system uses very similar components to the one developed by Quijano et al., but with a few differences, result of both design consideration, parts availability and toolkit requirements . For starters, to improve the system’s sensitivity, it was decided to take out the gRNA from the operon and control its expression by a constitutive promoter independent of the sporulation cascade, ensuring large presence of gRNA at the time of Cas9 activation, and only one gRNA was included in the final design. Moreover, sporulation-induced promoter PsspB was used instead of PsspA, as readily available in the Ellis’ Toolkit. The cloning method of choice was Golden Gate, in accordance with the B.subtilis toolkit. Similarly to the chitinase CDS, Cas9 CDS, due to its significant length, was ordered from IDT as two separate gBlock fragments, D15 and the gRNA, instead, were ordered as a single gBlock fragment. Cas9 CDS fragments were again designed with overhangs featuring BbsiI recognition sites and directional assembly, allowing for the reconstitution of the full Cas9 CDS into a L0 assembly.

The various L0 parts were subsequently assembled into 4 different L1 constructs. ‍

Figure 1. The scheme of our final self-digesting assmebly design

Our Experiment Result

Unfortunately, Due to time constraints, further progress was not made in assembling a self-digesting plasmid ready for expression in B. subtilis.

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