Part:BBa_K4304007
Cas12a-opt Plasmid
Cas12a-opt Plasmid (pET28a-FnCas12a) - BBa_K4304007
Contribution By Team PINGHE
Group: PINGHE iGEM 2023
Summary:
In the course of our research, we used the plasmid pET28a-FnCas12a (BBa_K4304007) for protein expression and purification, and performed cleavage experiments with the obtained Cas12a protein. We designed an assay system for the detection of S. aureus as well as Pseudomonas aeruginosa contamination in cosmetics (or other liquid solutions/semi-solids). The principle of this detection system is to use Cas12a protein to cut specific nucleic acid sequences of bacteria as a way to detect the presence of bacteria.
Characterization/Measurement
Cas-12a Protein Expression and Purification
We transformed the pET-28a(+)-LbCas12a-6His expression plasmid into E. coli BL21(DE3) and cultured overnight. Then we transferred the medium into 300mL fresh LB culture medium. When the OD600 was around 0.6, we added IPTG to induce LbCas12a expression at 16℃ for 12 hours. Then LbCas12a was extracted and purified by His-tag.
As shown in Figure 1, the size of the LbCas12a protein is between 100kDa and 160kDa. Hence we can see that the protein was successfully induced to express.
Figure 1: SDS-PAGE analysis of LbCas12a protein.
Function Validation
Figure 2: Graph of efficiency comparison of sgRNAs.
After testing the fluorescence of the sgRNA after incubation, we drew this graph to show the efficiency of the different sgRNAs. In the graph, a faster decreasing slope means higher efficiency in uncoiling the plasmid. Since all the sgRNA shows a decreasing slope, all of them successfully react with the plasmids, which means our experiment succeeded. The gbc-sg1RNA and the fem-sg3 decreased fastest with the highest change in slope among the same types, which means they have the highest efficiency.
Besides, we tested the reaction of the sgRNA in different concentrations of bacteria liquid containing plasmids pUC57-femA and pUC57-GbcA. By this, we can find the minimum detectable concentration and get the sensitivity of our test kit.
Figure 3: Relative fluorescence intensity of different sgRNA for femA (represent the sensitivity of different sgRNA).
Figure 4: Relative fluorescence intensity of different sgRNA for GbcA (represent the sensitivity of different sgRNA).
At the point that relative fluorescence intensity gets 1000000, human eyes could identify the change in the light intensity. Therefore, the line of relative fluorescence intensity of different sgRNA that gets the 1000000 in the least time would represent the most effective sgRNA. And by analyzing the 1ul and 2ul, we can find the sensitivity of each sgRNA. After analyzing, we find out that sgRNA1 in femA group and the sgRNA2 in GbcA group is the most sensitive sgRNA.
In conclusion, each sgRNA has their own advantages and disadvantages. Therefore, we would not focus on any single type of sgRNA to make our product. We will try to mix these sgRNA together in a specific concentration ratio to get the highest efficiency and sensitivity in detecting the pathogen.
Reference
- Buddenborg C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.
- Vento J.M., Crook N., Beisel C.L. Barriers to genome editing with CRISPR in bacteria [J]. J Ind Microbiol Biotechnol, 2019, 46(9-10): 1327-1341.
- Li Q., Sun M., Lv L., et al. Improving the editing efficiency of CRISPR-Cas9 by reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023, 12(3): 672-680.
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).
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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