Part:BBa_K4212045

SDP2

L1

Introduction to this part

This part contains PsspB promoter, RBS1, D15 CDS, 0D spacer.

Our Experiment Design

The core activity of our self-digesting circuit lies in the synergistic action of exonuclease D15 and DNA cutting capabilities of a CRISPR/Cas9 system, structurally (and functionally) joined together in a synthetic operon. In terms of actual construct design, plasmids 1B and 1C are devised to contain parts of the Cas9-D15 operon, later to be merged together with a Level 2 assembly in the destination plasmid. In particular, 1B contains the sporulation-induced promoter PsspB, a strong RBS (RBS1), D15’s coding sequence and a spacer, 0D. In this case, spacer 0D replaces the terminator slot, allowing construct 1B to assemble properly. Importantly, a D15 CDS linear fragment with standard overhangs for the Subtitoolkit was ordered from IDT; however, this contained a 3’ overhang tailored to terminator sequences which would fail to yield an orderly operon assembly. Hence, primers were designed to amplify D15 and introduce a new, purpose-built spacer overhang to ensure orderly assembly in the operon design (D15-1B)A schematic is presented below. High-fidelity PCR (Phusion) was carried out on 10 ng of linear D15 fragment with the aim of amplifying and extracting the D15 sequence with a spacer overhang tailored to the synthetic operon.

Figure 1. The scheme of our D15 assmebly design

Our Experiment Result

‍ Subsequently, a level 0 golden gate reaction was set up to allow entry of the D15-1B sequence into the toolkit part collection. The assembly was then transformed in E. coli,plated onto LB Cm and colonies were screened via cPCR (Phire - Thermofisher). Positive clones were sent for Sanger sequencing, and unmutated constructs were selected for culture collection deposit in a glycerol stock. Once in the part repository, it was time to transition to a Level 1 assembly bringing together the first part of the synthetic operon. Hence, adopting a similar workflow as for other assemblies, a L1 golden gate was set up, and successful assemblies were identified via transformation into e. coli and screening with Phire.

Figure 2. The result of our D15 assmebly design

References

[1] https://parts.igem.org/Part:BBa_K2232020

[2] Moyer, R.W. & Rothe, C.T. (1977) Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA. Journal of Virology. 24 (1), 177–193. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC515921/.

[3] https://goldengate.neb.com/#!/help

[4] 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
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 150
  Illegal PstI site found at 910
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 91
  Illegal PstI site found at 910
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 150
  Illegal PstI site found at 910
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 150
  Illegal PstI site found at 910
  Illegal NgoMIV site found at 66
  
• 1000
  COMPATIBLE WITH RFC[1000]