Team:Groningen/Design
DESIGN
Design
Introduction
Bacteria with
hCas9
Biobrick construction
To make the hCas9 operon biobick compatible we had to introduce the I473F mutation [Heler (2017)], remove the prohibited restriction sites 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). All fragments put together using gibson assembly. FigureTo create the hCas9 operon a combination of synthetic DNA and PCR fragments were used.
All DNA fragments had to be combined by Gibson assembly. Because we wanted to combine the fragments using Gibson assembly, all fragments had to be designed with a specific overhang. The fragments that did not need any modification were amplified from the original Cas9 operon on the pWJ40 plasmid. We first tried to PCR it from Streptococcus poygenes genomic DNA. Unfortunately this did not work. To solve this we used plasmids that contained the Cas9 operon(addgene).
Validation construction
CRISPR array
Biobrick construction
dCas9
Biobrick construction
We started out by trying to transform the dCas9 from the biobrick dCas9-Ω submission (part BBa_K1723000) into competent Escherichia coli DH5alpha cells, since the part is already in biobrick format and only requires removal of the Ω-subunit (link to primer design). 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 we PCR amplified the dCas9 out of the plasmid using primers .. & … In this PCR reaction a XbaI as well as a PstI site restriction site were incorporated in front of and behind dCas9, enabling dCas9 integration into an iGEM Vector (link) via restriction ligation (protocol link). 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).
To change the PAM, four mutations were required (see table). 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 insure 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 (part page") and dCas9-VRER (part page) were submitted to the iGEM HQ. We ordered two fragments of synthetic DNA(gBlocks) to introduce the I473F mutation and make the hCas9 operon biobrick compatible. To make the ......
The fragments were designed in such a way that they already contained the overhangs for the Gibson assembly. The first gBlock was used to remove the EcorI site and introduce the I473F mutation. The other gBlock was used to remove the ......
The other 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 transformed product was linearized using PCR
After the restriction ligation it was transformed into E. coli dh5α and colonies were picked to grow overnight. The overnight cultures were miniprepped to isolate the plasmids from the cells. The plasmid had to be linearized using PCR before it could be put together with the other fragments.
To combine all the fragments that we had created we used a Gibson assembly. We transformed the product into E. coli dh5α. We isolated the plasmids and performed several PCR reactions with different primers to confirm that it contained the desired product. We send the plasmids for sequencing further verification. Unfortunately it turned out that the gblock that we used for the assembly was not synthesized correctly. It did not contain the correct sequence, however it still had the expected size that is why we did not catch it earlier when we checked it on gel. Due to time constraints, because of the parts submission deadline, we were not able to order the gblock again and repeat the construction of the biobrick compatible hCas9.
So we were not able to construct a biobrick compatible hCas9 in time for the parts submission deadline. However, it would still be interesting for our project to see if hCas9 would work when expressed in L. lactis or E. coli. To do this we isolated the hCas9 operon including the repeats from S. pyogenes plasmid pRH180. For comparisson we also isolated the regular Cas9 operon from the pWJ40 plasmid. The vectors that we used are pBAD, an arabinose inducable E. coli expression vector, and pNZ8048, a nisin inducable L. lactis expression vector. We used overhang PCR to get the correct overhangs for the insert and the linearized vectors for the gibson assembly.
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.(protocol) 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 phages and the surviving colonies isolated. Using PCR we could than have measured the increase in size of the spacer array. The amount of surviving colonies and the sizes of the spacer array’s would have given an indication about the efficiency/activity of the hCas9 operon in L. lactis.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 amplified 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
References
- Kuipers OP, de Ruyter PG, Kleerebezem M, de Vos WM. Journal of Biotechnology. 1998;64:15–21
- van de Guchte, M., van der Vossen, J.M.B.M., Kok, J. and Venema, G. (1989) Appl. Environ. Microbiol. 55, 224-228
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