Part:BBa_K4611009
His-SUMO-LwaCas13a with lacI
This part contains the coding sequences for lacI protein and LwaCas13a for synthesizing LwaCas13a and subjecting it to regulation by IPTG, which is regulated by the lacI promoter for lacI deterrent proteins and by the T7 promoter for LwaCas13a.
Usage and Biology
Experiment
In order to obtain a plasmid encoding LwaCas13a protein with pSB1C3 as the plasmid backbone, we used two plasmids, pveg and pC013-Twinstrep-SUMO-huLwCas13a, as templates for PCR. All PCR steps followed the instructions of the PrimeSTAR® HS DNA Polymerase(Takara) kit, using the 3-step method.
Plasmid construction:
PCR
- Backbone F primer and Backbone R primer were used for PCR amplification using pveg plasmid as template
- LacI F primer and LacI R primer were used for PCR amplification using pC013-Twinstrep-SUMO-huLwCas13a plasmid as template
- Cas F primer and Cas R primer were used for PCR amplification using pC013-Twinstrep-SUMO-huLwCas13a plasmid as template
Through gel electrophoresis experiments we proved that the PCR experiments successfully amplified the DNA sequences ready for the Golden Gate assembly.
Figure 1: Gel electrophoresis of the PCR product for Golden Gate assembly of pSB1C3-His-SUMO-LwaCas13a plasmid. Lane1: DNA marker. Lane2-Lane3: LwaCas13a. Lane4-Lane5: LacI operating system. Lane6-Lane7: pSB1C3 Backbone.
Golden Gate Assembly:
- Golden Gate Assembly: Golden Gate Assembly was carried out using the above three groups of PCR amplification products.
- DNA extraction and purification: After assembly, the assembly product was used for plasmid transformation, and the LB solid medium with chloramphenicol was used for colony screening.
- Colony PCR: The colony was confirmed by colony PCR to verify the success of Golden Gate assembly and plasmid transformation.
Figure 2 Gel electrophoresis of the colony PCR product. (A) Illustration of PCR Product 1. (B) Illustration of PCR Product 2. (C) Lane1: Marker. Lane2-Lane4: The product amplified by Primer VR and Primer CHECK F. Lane5-Lane7: The product amplified by Primer CHECK R and Primer VF2.
4. Enzymatic validation: The plasmid was extracted from the transformed colony, and the product of plasmid extraction was digested with enzyme to further verify the success of assembly and plasmid transformation.
Figure 3 (A) Digestion using EcoRI enzymes results in a band with a length of 7780bp; (B) Digestion using EcoRI and SpeI results in two bands. One is 5712 bp, and the other is 2068 bp. (C) Lane1: Marker. Lane2: Plasmid was digested with EcoRI and SpeI. Lane3: Plasmid was digested with EcoRI.
The final plasmid used pSB1C3 as the plasmid backbone and contained Biobrick prefix and suffix, which can be connected to other components by 3A assembly method, such as constructing crRNA-Cas13a co-expression system.
Expression and purification of LwaCas13a protein
Five 15mL tubes were taken, 5 mL LB medium and 1 mL of bacteria-glycerol mixture were added to each tube, and cultured on a shaker for 4 h until the bacteria were cloudy. They were then cultured in 1L flasks, each vial was supplemented with 250mL of medium and 250uL of CmR solution, and incubated well for 8 h. Collect 1mL of bacterial solution for later use. Subsequently, 200 μL of IPTG was added to each vial and incubated overnight at 27 °C.
Trans1-T1 bacteria with pSB1C3-His-SUMO-LwaCas13a plasmid were cultured, and IPTG was added to induce the expression of LwaCas13a protein. The cell lysate from bacteria before and after induction were analyzed by SDS-PAGE gel electrophoresis, and the results indicated that expression of LwaCas13a was induced successfully (Figure 4).
Figure 4 Results of electrophoresis of cell lysate from bacteria before and after induction. Lane1:Protein marker. Lane2-Lane6: Uninduced cell lysate from bacteria. Lane7-Lane11: IPTG-induced cell lysate from bacteria.
Bacteria were lysed by ultrasound, and the cell lysate is then centrifuged. The soluble protein will be present in the supernatant and the insoluble component in the pellet. and both supernatant and pellet were analyzed by SDS-PAGE (Figure 5). The electrophoretic results showed that the amount of LwaCas13a protein in the precipitation was very little, and most of LwaCas13a protein was in the supernatant, indicating that SUMO tagged LwaCas13a was soluble.
Figure 5 Perform SDS-PAGE detection of lysed supernatant of cells with lysis pellet of cells. Lane1:Protein marker. Lane2-Lane6: Lysed supernatant of cells. Lane7-Lane11: Lysis pellet of cells.
Western blot examined uninduced bacteria for RIPA-lysed, induced bacteria for RIPA-lysis, and induced bacteria for ultrasonic lysis showed that SUMO-LwaCas13a protein had been expressed successfully (Figure 6).
Figure 6 Western Blot with LwaCas13a antibody. Lane1: Protein marker. Lane2-Lane3: Uninduced bacteria for RIPA-lysed. Lane4-Lane5: Induced bacteria for RIPA-lysis. Lane6-Lane7: Induced bacteria for ultrasonic lysis.
The final step of LwaCas13a production was to use His-Tag Protein Purification Kit with IDA-Ni Magnetic Agarose Beads for purification and SUMO protease ULP1 to digest SUMO-LwaCas13a. We use SDS-PAGE gel electrophoresis to analyze the purification results (Figure 7).
Figure 7 Results of purification of LwaCas13a protein. Lane1:Protein marker. Lane2-Lane3: Uninduced bacteria for RIPA-lysed. Lane4-Lane5: Induced bacteria for RIPA-lysis. Lane6-Lane7: Induced bacteria for ultrasonic lysis. Lane8-Lane9: Purified LwaCas13a protein.
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Illegal PstI site found at 4404 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1082
Illegal EcoRI site found at 4383
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Illegal BglII site found at 4081 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1082
Illegal EcoRI site found at 4383
Illegal PstI site found at 4404 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1082
Illegal EcoRI site found at 4383
Illegal PstI site found at 4404 - 1000COMPATIBLE WITH RFC[1000]
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