Part:BBa_K4304007
Cas12a-opt Plasmid
Cas12a Plasmid
Contribution
The CRISPR-Cas12 system recognizes the PAM-containing dsDNA under the guidance of the guide RNA, and then promotes the unwinding of the target dsDNA, and the target strand (TS) in the unwrapped target dsDNA forms an Rloop with the guide RNA, thereby releasing the RuvC in Cas12. The non-target strand (NTS) in the target dsDNA just after melting will be cleaved by RuvC of the released active site; the result of this cleavage causes the unwinding of the target DNA, releasing the The TS of the target dsDNA is cleaved by RuvC; when Cas12 completes the cleavage of TS and NTS in the target dsDNA (cis cleavage), the dsDNA will be released, and the active site of RuvC, which is left in space at this time, once there is a When ssDNA enters, it will be cut (trans-cut).
Based on CRISPR pathogenic microbial detection technology was developed in recent years, and the DETECTR system, which is based on CRISPR-Cas12a, was applied for pathogenic microorganism detection. In order to verify if there are related parts, we searched the iGEM Biological Parts library and picked BBa_K2644101. This is a biological part submitted by iGEM18_TJU_China in 2018, and the team provided the DNA sequence of FnCas12a, and they measured the effect of ions on FnCas12a’s cleavage activity. Our team developed a reaction platform to detect pathogenic microorganisms, such as salmonella and shigella, adding data from in vitro DETECTR reaction system. This information can be a good reference for future iGEM teams working on in vitro DETECTR reaction systems.
The gene fragments ipaH and invA are amplified from salmonella and shigella genomic DNA, we synthesized these DNA fragments and insert them in the pUC57 vector. Next, we mixed the FnCas12a protein with sgRNA corresponding to the two genes, after they formed a complex, then we added these plasmids and buffer. Finally, we did gel electrophoresis to verify if the in vitro reaction system worked well.
Engineering Success
Construction of plasmids
We designed the plasmids: the FnCas12 protein expression plasmid, In order to construct our plasmids, we let the company synthesize the DNA fragments, FnCas12 was inserted into the pET28a vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB solid medium plates containing corresponding antibiotics, and incubate them at 37℃ overnight.
Expression and purification of Cas12a protein
We transformed the pET28a-FnCas12a expression plasmid into E. coli BL21(DE3) competent cells, and cultured at 37℃ overnight (Figure 1A). we inoculated a single colony into LB (Kana+) culture medium, incubated overnight, and then transferred the cultured medium into 1L fresh LB (Kana+) culture medium. We induced the expression of FnCas12a with IPTG when the OD600 was around 0.6-1.0, and cultured at 16℃ for 12h. Subsequently, we used nickel affinity purification to purify the acquired Cas12a proteins from other proteins in E. coli (Figure 1B).
A. Incubate the plasmid pET28a-FnCas12a containing BL21(DE3). B. SDS-PAGE electrophoresis gel of Cas12a protein compared to nonspecific protein impurities.
Bicinchoninic Acid Assay (BCA)
Cas12a protein has a size of 130kDa. The SDS-PAGE electrophoresis result indicates that the Cas12a protein is present in the solution we collected at 130kDa, and not present in the nonspecific protein impurities. Thus, Cas12a proteins were expressed and purified with high quality. Then, we tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA), using SpectraMax i3x Multi-Mode Microplate Reader with the absorption peak at 562nm (Figure 2).
Table 1. Absorbance and calculated protein concentration of Cas12a 1 and Cas12a 2.
With this BCA standard curve, we measured the concentration of two samples of Cas12a protein, they are 10.9 µg/mL and 7.32 µg/mL respectively. This result indicated that we obtained a sufficient concentration of Cas12a protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1591
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 365
Illegal BglII site found at 1544
Illegal BglII site found at 1578
Illegal BglII site found at 1623
Illegal BglII site found at 2402
Illegal BglII site found at 3383
Illegal BglII site found at 8344 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 6564
Illegal NgoMIV site found at 6724
Illegal NgoMIV site found at 8312
Illegal AgeI site found at 3184
Illegal AgeI site found at 3253 - 1000COMPATIBLE WITH RFC[1000]
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