Design

Introduction

Bacteria with

hCas9

Biobrick construction

As a basis for the biobrick compatible hCas9 operon we used the Cas9 CRISPR operon from Streptococcus pyogenes. To make this into a biobrick compatible hCas9 operon we had to introduce the I473F mutation (Heler et al.), remove the prohibited restriction sites it contained and attach the biobrick prefix and suffix.

To achieve this we used a combination of synthetic DNA (gBlocks) and DNA fragments that were PCR amplified from pWJ40 (containing the Cas9 operon), which our supervisor Chenxi gave us. All fragments were finally put together using Gibson Assembly. All cloning steps were performed in E. coli DH5α. Figure 1 gives an overview of all the cloning steps that were used. Besides the hCas9 operon the tracer RNA and the spacer array are also required to get a successful CRISPR response. In this project we constructed biobrick compatible tracer RNA and several pre-programmed spacers arrays.

• The first gblock was used to remove the XbaI site and introduce the iGEM prefix and suffix. This part was first placed in the pSB1A3 vector to form the backbone for the Gibson assembly. The plasmid had to be linearized using PCR before it could be put together with the other fragments.
• The second gblock was used to introduce the I473F mutation and remove the EcorI site.
• The PCR fragments that were amplified from the Heler plasmid were designed in such a way that they already contained the overhangs for the Gibson assembly.

To combine all the fragments that we created we used a Gibson assembly. After transformation the product into E. coli DH5α and isolation of the plasmid, several PCR reactions were performed with different primers to confirm that it contained the desired product. After the gels seemed to be correct we sent the plasmid for sequencing and further verification. Unfortunately, it turned out that the gblock containing the I473F mutation used for the assembly was not synthesized correctly. It did not contain the correct sequence, however it still had the expected size which is why we did not catch it earlier when we checked it on gel. Due to time constraints of the parts submission deadline, we were not able to order the gblock again and repeat the construction of the biobrick compatible hCas9. We encourage next iGEM teams to pick up where we left as hCas9 seems to be a valuable addition to the iGEM registry!



Validation construction

As mentioned earlier we were not able to construct the biobrick compatible hCas9 operon. So the experiment that will be described next was not executed. To validate that hCas9 is capable of acquiring spacers in both L. lactis and E.coli, we wanted to perform an on-plate acquisition assay. In this assay we would have measured the rate at which spacers would be acquired. For this assay we first would have inserted the hCas9 operon into an inducible expression vector. We were planning to use an arabinose inducible pBad vector for E. coli and a nisin inducible pNZ8048 vector for L. lactis. For comparison we would also have included the regular Cas9 operon. The cells would then be exposed to bacteriophages and the surviving colonies isolated. Using PCR we could then have measured the increase in size of the spacer array. The amount of surviving colonies and the sizes of the spacer arrays would have given an indication about the efficiency/activity of the hCas9 operon in L. lactis and E.coli.

CRISPR array

Biobrick construction

dCas9

Biobrick construction

For the next part for our project we created a biobrick compatible dCas9 protein that would recognize different PAM sites than hCas9. To achieve this we had to introduce the VRER mutations into the dCas9 gene,(Table) that were described by Heler et al. We started out by trying to transform the dCas9 from the biobrick dCas9-Ω submission (part BBa_K1723000)which was provided in the iGEM distribution plate, as a basis. However, we were not able to recover successful transformants. Therefore we decided to pursue another strategy utilizing the addgene plasmid pJWV102-PL-dCas9 (link to benchling), which was supplied to us by our supervisor Chenxi.

• To start off the dCas9 was PCR amplified out of the plasmid using primers to incorporate XbaI as well as a PstI restriction sites in front of and behind dCas9, enabling dCas9 integration into an iGEM Vector.
• Next we removed the EcoRI site, since this was still present in the Addgene plasmid and interfering with Biobrick compatibility. In order to accomplish this, two sets of quick-change primers were designed (image of sequence dCas9 plasmid part containing EcoRI site and all four primers). This created the regular dCas9, which can be seen as an improvement on (BBa_K1026000).
• Since all mutations are positioned at the end of the gene, a gBlock was designed containing the end of dCas9 with all four mutations. To exchange the gblock with the original end of dCas9 the BamHI restriction site was used. The end of the gblock contained the biobrick suffix. To ensure that only mutated dCas9 would be transformed, the correct fragment was subjected to a gel-extraction after restriction of the pSB1C3 dCas9 plasmid.
From this sub-project both the biobrick-compatible dCas9 (BBa_K23610000) and dCas9-VRER (BBa_K2361001) were submitted to the iGEM HQ.

Validation

To validate that dCas9 worked we designed an experiment in which we would be able to determine if dCas9 was capable of interfering with the expression of GFP. Since the spacer array and the tracerRNA are not on the same plasmid we used pre-designed guide RNA’s. The guide RNA’s were designed so they would bind in the coding sequence of GFP after a constitutive promoter. We did not only want to see if our dCas9 could interfere with gene expression, we also wanted to see if the VRER mutations would cause it to recognize a different PAM sequence. So half of the guideRNA’s had a PAM recognition sequence for dCas9 and the other half had the recognition sequence for dCas9VRER. This way we could compare the relative amount of GFP inhibition between the guide RNA’s and determine of the VRER mutations changed the recognition sequence for dCas9.

Target array / reporter

Biobrick construction




Validation construction

Lactis toolbox

Biobrick construction

As our detection system is designed to ultimately by integrated into L. lactis, we wanted to provide the registry with the desired promoters which were not available in a pSB1C3 backbone. For each promoter, a different plasmid and primer pair was used to amplify the sequences from their native backbones. The pNisA promoter was amplified from the pNZ8048 plasmid using the G65 and G66 primers [2]. The p32 promoter was amplified from the pMG36E plasmid using the G67 and G68 primers[7]. The pUsp45 promoter was amplified from the already cloned part BBa_K2361003 using the G63 and G64 primers. This added the biobrick restriction sites combinations to the flanks of the promoter sequence. This allowed us to incorporate the promoter sequences into the biobrick-compatible format.


Validation construction

So how do we actually validate our promotors? Since iGEM requires biobrick submissions to be contained in the PSB1C3 vector, the first step was cloning the promotors in the lactis expression vector (pNZ8048) together with an sfGFP molecule. This was then transformed into Lactis. Furthermore the promotor strength was classified by measuring GFP expression. For the inducible promotor Pnis we used several different Nisin concentrations to estimate the effect on expression strength. Since the sequencing data for p32 & push45 (links???) did not match the expected sequence we resorted to only testing Pnis activity. See link for the results.

References

2. Kuipers OP, de Ruyter PG, Kleerebezem M, de Vos WM. Journal of Biotechnology. 1998;64:15–21
7. van de Guchte, M., van der Vossen, J.M.B.M., Kok, J. and Venema, G. (1989) Appl. Environ. Microbiol. 55, 224-228

What should this page contain?

• Explanation of the engineering principles your team used in your design
• Discussion of the design iterations your team went through
• Experimental plan to test your designs

Inspiration

• 2016 MIT
• 2016 BostonU
• 2016 NCTU Formosa

Next: Results