gRNA Level 1 assembly

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).

Figure 1: the level 1 assembly of gRNA, constructing L1A for the final L2 construct

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 distinguish the gRNA from primer dimers, but a reduced primer concentration helped us identify correct clones.

Figure 2: the colony PCR gel image to verify the L0 gRNA plasmid in the transformants. The length of the correct band is 117bp, amplified by pair of verification primers: oSP013 and oSP015.

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.

Figure 3: the colony PCR gel image to verify whether the L0 region is transformed the L1 gRNA plasmid, amplified by pair of verification primers: oSP013 and oSP015.

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

Figure 4: restriction digestion of L1 gRNA construct using BpiI

D15 Level 1 assembly

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).

Figure 5: the level 1 assembly of D15, constructing L1B for the final L2 construct

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 6: amplification of D15 with the addition of overhangs to facilitate cloning into the 1B vector.

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).

Figure 7: the colony PCR gel image to verigy wether the linear fragment of D15 previously amplified was successfully inserted into a L0 vector.

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. Finally, a restriction digest was performed to confirm the presence of the insert.

Figure 8: the colony PCR gel image to verify whether the L0 region is transformed the L1 D15 plasmid, amplified by pair of verification primers: oSP013 and oSP014.

Figure 9: restriction digestion of L1 D15 construct using BsmbI

Cas9 Level 1 assembly

Cas9 is a large protein, consisting of 1368 residues ( ≈4.1kb). Its synthesis from IDT as a single linear fragment was ruled out due to the complexity of the build. For this reason, we once again relied on customized overhangs which split the protein CDS’s in half to allow for a directional and scarless assembly. In particular, these would come together and reconstitute the CDS in a L0 vector (STK001). Then, by harnessing the destination backbone 1C and 0A spacer, Cas9 would be paired with the appropriate regulatory elements (RBS and terminator) to allow for its translation. Indeed, Cas9’s construction in 1C lacks a promoter as the transcriptional levels are regulated upstream by PsspB (1B vector).

Figure 10: the level 1 assembly of Cas9, constructing L1A for the final L2 construct

Notably, cas9 was the largest protein we attempted to clone across Sporadicate. As soon as linear fragments were obtained and resuspended at 50fmol/μL in water, we tested two different molar ratios of vector to insert (1:5 and 1:3) in L0 Golden Gate assemblies. This choice is motivated by the attempt to drive assembly of Cas9 in the destination backbone (1C) by enhancing the pool of CDS inserts in the reaction mix. However, following transformation in E. coli and recovery on LB Amp, only incorrect colonies were present at the 16h time point.

Figure 11: the colony PCR gel image to verify the L0 Cas9 plasmid in the transformants. The length of the correct band is 1363bp, amplified by pair of verification primers: oSP033 and oSP034.

Considering results obtained at this stage, it became apparent that our initial strategy to clone Cas9 in STK001 was not optimal. Hence, we asked for guidance to our supervisors Mo and Joaquin, who recommended decreasing the molar ratio to 2:1 while increasing cutting times with BsmBI (from 3min to 5min) and the number of reaction cycles (from 30x to 60x). Indeed, due to the size of the final product, longer digestion phases coupled with more reaction cycles may have allowed for a more efficient golden gate cloning process.

Subsequently, assemblies were set up and transformed in E. coli TOP10, to then being left to recover at 37C overnight. The following day, we noticed that no colonies grew, but we still decided to give the cells a larger time window to allow for growth given the large insert size. After the 20h mark from the initial transformation, a large number of colonies appeared. At that point, adhering to the cloning framework adopted across the summer, colonies were screened via colony PCR to shortlist positive clones. Positive hits were isolated and grown overnight to be miniprepped and sent for sequencing. Correct constructs were then cultivated for preparation of glycerol stocks.

Figure 12: the colony PCR gel image to verify whether the Cas9 linear fragments are assembled into the L0 vector.

Having successfully assembled and secured Cas9’s CDS in the toolkit, it was now time to construct the final module of the D15-Cas9 operon (i.e., 1C). In particular, construct 1C starts with spacer 0A (replacing this time the promoter slot) and is followed by RBS optRBS, the full Cas9 CDS, and terminator B0015. To that end, a higher order assembly was performed utilizing pCas9 (L0) c7, leading to colonies of 1C for screening. Due to the big size of the construct, colonies were screened via restriction digest analysis of the assembled plasmid; correct assemblies were stored in 50% glycerol (1:1) at -80C.

Figure 13: the restriction digest gel image of the L0 Cas9 clones with BsaI.

Reporter Level 1 assembly

With 1A, 1B, and 1C successfully assembled and secured in glycerol stocks, the final step in allowing validation of our biocontrol measure lied in the construction of a reporter system to quantify the efficacy of the self-digesting circuitry. The approach adopted by Quijano et al. involves an anchor protein-GFP fusion to assess protein displaying abilities as well as plasmid retention following induction of sporulation. The authors used colony PCR combined with microscopy techniques to quantify effective protein display as well as plasmid retention post-germination.
Initially, we set out to follow the methodology outlined above, i.e. harnessing a fluorescent fusion protein to investigate the efficacy and impact of plasmid self digestion with our adapted circuitry. To that end, we designed primers for the amplification of GFPmut3b from STK053 in the STK toolkit, to be joined with the CotG linear fragment amplified as part of our Chitinase Display strategy in a Golden Gate reaction.

Figure 14: PCR amplification of GFPmut3b from STK053 containing GFPmut3b CDS. The pair of primers used for amplification is oSP024 and oSP025, the correct length is 753bp.

However, as we tried multiple assembly runs, CotG-GFPmut3b (L0) fusions could not be obtained. Notably, different troubleshooting techniques we adopted across our experience with STK cloning were tested. This includes modifying number of cycles, molar ratios, length of digestion and ligation steps in our Golden Gate protocol, and many others.
Hence, we revisited our design to circumvent this issue. Instead of fusing GFP to an anchor protein, we decided to harness constitutive GFPmut3b (a GFP coding sequence optimized for expression in B.subtilis) expression in vegetative cells as a proxy for successful plasmid degradation. Indeed, if plasmids exhibited self-digesting activity, spores generated from successful transformants would not contain any DNA code to express GFPmut3b. Meaning, once germination is induced and the spores turn to cells, they should no longer present fluorescence. Consequently, our new revisited pipeline would still involve assembly of a self digesting circuit, and evaluation of its efficacy using a reporter gene. In the interest of time, we opted to utilize a GFPmut3b transcriptional unit present in the toolkit, featuring constitutive promoter STK034, RBS sequence RBS_3, GFPmut3b CDS and terminator t13s3p43. However, this L1 plasmid consisted of a 1B backbone, meaning we could not integrate it into our L2 EXP plasmid. Thus, we opted to continue our design in a L2A plasmid, rather than L2 EXP, completing it with a 1D spacer, whilst also assembling a L2B plasmid with the L1B GFPmut3B TU and spacers in place of L1A, L1C and L1D. These two L2 plasmids could then be assembled into an expression vector (STK109), using the BsaI restriction enzyme.

Figure 15: the colony PCR gel image to verify whether the L2A and L2B constructs are successfully obtained.

Within the course of the project, the L2A plasmid was successfully assembled as noted in figure xy which displays a cPCR result and restriction digest of positive colonies from the same. However, the L2B plasmid containing the GFPmut3B TU was not achieved, even after subsequent troubleshooting attempts. Due to time constraints, further progress was not made in assembling a self-digesting plasmid ready for expression in B. subtilis.