Difference between revisions of "Part:BBa K4223008"

Revision as of 06:27, 25 September 2022

Cas14a1 protein expression system

CRISPR-Cas14 protein, which is found almost exclusively in the superphylum of Archaeophilus. CRISPR-Cas14 protein has the activity of targeting single stranded ssDNA, and it is twice smaller than CRISPR-Cas9 protein.
Combining the nonspecific ssDNase cleavage activity of CRISPR Cas14 protein with an isothermal amplification method (DETECTR-Cas14), it is also promising to be developed for high-fidelity DNA single nucleotide polymorphism genotyping to detect ssDNA viruses, with potential clinical, ecological, and economic importance. Thus, CRISPR-Cas14 could play a huge role in CRISPR diagnostics for both infectious and noncommunicable diseases.
Based on previous research, we developed a single-base, amplification-free, portable nucleic acid detection platform based on Cas14a1 with high fidelity, high specificity, and high efficiency.

Protein Expression and Purification

Solution required for the experiment<h3/> (1) LB (Luria-Bertani) liquid medium.
1% (w/v) Tryptone, 1% (w/v) NaCl, 0.5% (w/v) Yeast Paste.
(2) TEV and Cas14a1 affinity chromatography (Ni column) purification solutions.
Lysis solution: 10 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5% glycerol, 5 mM β-mercaptoethanol.
Equilibration solution: 0 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
Elution solution: 300 mM imidazole, 500 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
(3) Cas14a1 heparin column purification solution.
Equilibrium solution: 100 mM Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.
Elution solution: 2 M Nacl, 20 mM Tris-Hcl, 5 mM β-mercaptoethanol.


<h3>Plasmid Transformation and Protein Induced Expression<h3/> Transformation of MBP-Cas14a1 plasmid into DE3 cells was performed as follows: DE3 receptor cells frozen at -80°C were removed and thawed naturally in an ice box (DE3 cells should be used once to avoid reuse after freezing), 1 μL of the above plasmid was aspirated and mixed homogenously with 50 μL of DE3, placed on ice for 30 min, transferred to 42°C for 90 sec and then immediately placed on ice for 3 min. Immediately place on ice for 3 min. add 700 μL LB medium (without antibiotics) and resuscitate for 1 hr at 37°C 180 rpm (all cultures were at this shaker speed and will not be repeated) under aseptic conditions. The successfully transformed E. coli was centrifuged at high speed, 700 μL supernatant was discarded and the remaining bacterial solution was mixed, and 30 μL was evenly coated with LB solid medium containing 50 μg/mL ampicillin (AMP) using a spreading rod and incubated in a light incubator at 37°C for 12 h. The monoclonal bacteria were then picked and cultured in 10 mL EP tubes, and the monoclonal strain was incubated overnight in 20% glycerol. After overnight culture, the monoclonal strains were stored in 20% glycerol for freezing.

Through literature review, we found that the induced expression of Cas14a1 increased and then decreased with the increase of isopropyl-β-D-thiogalactoside (IPTG) concentration, and the expression was higher at 0.2 mM IPTG, and the best induction was achieved at the temperature of 18°C.

Figure 1. SDS-PAGE to detect the expression of Cas14a1 under different inducer concentration (a) and different induction temperature (b)

Therefore, we added glycerobacteria containing Cas14a1 plasmid at a final concentration of 1% to 10 mL EP tubes containing AMP and LB medium for a small amount of propagation culture, and 12 h later transferred fresh bacterial solution from EP tubes at the same 1% in 1000 mL conical flasks at 37°C, 180 rpm, and carried out a large amount of propagation culture until OD600 was 0.6. 10His-MBP- Cas14a1 protein was expressed at 18°C, 180 rpm, and induced overnight by adding 0.2 mM IPTG to a final concentration of 0.2 M.

<h3>Cas14a1 protein purification by affinity chromatography (Ni column)<h3/> Cells were centrifuged at 10,000 rpm for 10 min at 4°C. The pellet was collected, resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 5% glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole), and broken up by ultrasonication. The temperature was lowered to 4°C, and cell pellets were removed by centrifugation at 8000 rpm and 30 min. The protein supernatant was added to Bio-Scale Mini Nuvia IMAC Ni - charge (Bio-Rad laboratories, Inc., USA) in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol buffer with increasing concentrations of imidazole (from 10 to 300 mM). eluted with a linear gradient (refer to the following content (7)).

Cas14a1 affinity chromatography (Ni column) purification: The Cas14a1 purification procedure is divided into 7 steps: cleaning, equilibration, sample loading, washing, elution, water washing and 20% ethanol filling of the column. (1) Washing the Ni column with pure water (A1 channel) at a flow rate of 7 mL/min for 10 volumes. (2) Equilibrating the Ni column with 98% equilibration solution (A2 channel) + 2% eluent (B channel) at a flow rate of 7 mL/min for 5 volumes. (3) Protein loading (spiked channel) at a flow rate of 1 mL/min for 120 mL. (4) Washing the Ni column with equilibration solution (A2 channel) for 5 volumes. (5) Gradient elution of target protein in eluent (B channel), flow rate 3 mL/min, 6 volume for 0-30% elution; 10 volume for 30-40% elution; 5 volume for 40%-60% elution; 5 volume for 60%-70% elution; 6 volume for 70%-100% elution; 6 volume for 100%-100% elution. 6 volume; 100%-100% elution 10 volume. (6) Pure water (A1 channel) washed the residual solution in the purification column with a flow rate of 7 mL/min and 6 volume. (7) 20% ethanol (A3 channel) cleaned the purification column with a flow rate of 5 mL/min and eluted 5 volume, filled with ethanol, unloaded the column and immersed in 20% ethanol, and stored at 4 ℃ for proper storage. The role of low temperature and 20% ethanol is to avoid the growth of bacteria, which affect the re-use.

The target protein was purified by using the protein His-tag to chelate with nickel in the affinity chromatography column to enrich the target protein, and then the protein was eluted by imidazole competition. The results of SDS-PAGE characterization of Cas14a1 are shown in Figure 2. The appearance of a single band near 108 kDa, consistent with the theoretical molecular weight of Cas14a1 (Figure 2a), indicates that the protein was successfully purified and ready for subsequent experiments.


Figure 2. SDS-PAGE detection Cas14a1 (a) Ni column purification
M: Protein Marker; 1-4: indicate the number of tubes for collecting Cas14a1 protein fractions.
<h3>TEV protease cleaved MBP tag<h3/> TEV enzyme was homogeneously mixed with Cas14a1 protein at a 1:1 concentration ratio and then digested at 4°C for 12 h. The 10×His- MBP label was removed for subsequent heparin column purification.
The digestion results were verified by SDS-PAGE gel electrophoresis assay. As shown in Figure 3, TEV was added to the fusion protein Mbp-Cas14a1 (108 kDa), and the theoretical size band at 108 kDa disappeared after 12 h, while the excised labeled Cas14a1 band appeared at 63 kDa, indicating that the enzymatic cleavage was relatively complete. The mixed proteins after digestion were continued to be purified by heparin column.

Figure 3. SDS-PAGE detection of TEV digestion MBP-Cas14a1: uncut Cas14a1 protein; +TEV: 12h after digestion; Marker: protein rainbow Marker. Sequence and Features

Assembly Compatibility:

• 10
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  Illegal EcoRI site found at 2555
  Illegal PstI site found at 1345
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• 12
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  Illegal EcoRI site found at 2555
  Illegal NheI site found at 2785
  Illegal PstI site found at 1345
  Illegal PstI site found at 1511
  
• 21
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  Illegal EcoRI site found at 2555
  Illegal BglII site found at 436
  Illegal BamHI site found at 2149
  
• 23
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  Illegal EcoRI site found at 2555
  Illegal PstI site found at 1345
  Illegal PstI site found at 1511
  
• 25
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  Illegal EcoRI site found at 2555
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• 1000
  COMPATIBLE WITH RFC[1000]