Part:BBa_K4959004
SgaMcrA-pET28a
SgaMcrA-pET28a is a novel plasmid constructed using the pET-28a vector and a gene fragment called SgaMcrA. Introduction of this recombinant plasmid into BL21 competent cell produces the protein we need, a sulfur-modification-dependent restriction enzyme. The protein produced enables us to conduct the next functional study experiments. We first performed double digestion of the pET28a vector and the target fragment to construct recombinant plasmids using two restriction endonucleases, Nde1 and Xho1. After digestion, we ran the gel by agarose gel electrophoresis, followed by gel recovery to purify the DNA to improve its purity. Finally, we ligated the vector backbone, and the target fragment with T4 DNA ligase transformed them into E. coli sensory state by heat-excited method, and cultured them on resistant plates overnight.
SgaMcrA-pET28a (BBa_K4959004)
Construction Design
SgaMcrA-pET28a is a novel plasmid constructed using the pET-28a vector (BBa_K3521004) and a gene fragment called SgaMcrA (BBa_K4959001). Introduction of this recombinant plasmid into BL21 competent cell produces the protein we need, a sulfur-modification-dependent restriction enzyme. The protein produced enables us to conduct the next functional study experiments.
We first performed double digestion of the pET28a vector and the target fragment to construct recombinant plasmids using two restriction endonucleases, Nde1 and Xho1. After digestion, we ran the gel by agarose gel electrophoresis, followed by gel recovery to purify the DNA to improve its purity. Finally, we ligated the vector backbone, and the target fragment with T4 DNA ligase transformed them into E. coli sensory state by heat-excited method, and cultured them on resistant plates overnight.
Experimental Procedure
We planned to construct the plasmid using an enzyme-conjugated method. First, we obtained the target sequence (synthesized by Bio) from the designer. Then we digested the vector and the target fragment with two enzymes, NdeI and XhoI, respectively. Finally, we used T4 DNA ligase to link the target fragment to the vector backbone and the target fragment.
After that, we transformed the recombinant plasmid into E. coli receptor cells and grew them overnight on Kana resistant plates. The next day, we verified the plasmid lifting and digestion of the strains grown on the plates to make sure we got the correct recombinant plasmid.
Protein Expression and Purification
After that, IPTG is added to induce protein expression. To get a pure target protein, we used Nickel column purification, and an SDS PAGE was done to show whether we have our target protein and whether it is purified or not.
a: protein from solution with elution buffer
b: protein from solution with wash buffer
c: protein from supernate from ultrasonication
The target protein solution was again concentrated, and then SDS PAGE was done.
Function Testing
After extracting the target proteins, purification (nickel affinity chromatography, Q column chromatography, gravity column) and concentration were done, preparing for two function analyses: EMSA and nucleic acid cleavage test. This part presents the overview and experiment results of the function test for the enzyme we obtained.
Electrophoretic Mobility Shift Assays (EMSA)
The EMSA test aims to test the binding specificity (phosphorothioate dependent in this case) of the Sga enzyme that is purified. EMSA 5x buffer is prepared with 100 mM Tris-Cl and 50 mM NaCl concentrations. A 10ul system is then used to achieve binding between the target enzyme and the dsDNA – phosphorothioate B7A and non-phosphorothioate BL21.
The dsDNA is prepared from annealing of given ssDNA. Enzyme binding is followed by SDS PAGE (Sodium dodecyl sulfate – polyacrylamide gel electrophoresis). The product obtained is then stained using SYBR Gold (Invitrogen) without light, thus observed using a gel imager. We expect enzyme binding with only ptDNA, thus no binding with non-ptDNA.
(A) Result of BL21 EMSA
(B) Result of B7A EMSA
Nucleic Acid Cleavage Test
The nucleic acid cleavage test aims to test the cleavage specificity (ptDNA dependent in this case) of the enzyme we obtained. Cleavage 2x buffer is prepared with 40mM Bis-Tris, 100mM NaCl, 2mM DTT, and 2mM MnCl2 concentrations.
Enzyme cleavage is followed by enzyme digestion. Protein K is used to digest the enzyme Sga, avoiding potential influence in the following agarose gel electrophoresis (AGE). The samples are run on AGE; the results are shown in Figure 7. We expect to see cleavage only on ptDNA while no successful cleavage on non-ptDNA.
Reference
[1] Liu G, Fu W, Zhang Z, He Y, Yu H, Wang Y, Wang X, Zhao YL, Deng Z, Wu G, He X. Structural basis for the recognition of sulfur in phosphorothioated DNA. Nat Commun. 2018 Nov 8;9(1):4689.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4554
Illegal EcoRI site found at 4971
Illegal PstI site found at 5523 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4554
Illegal EcoRI site found at 4971
Illegal PstI site found at 5523 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4554
Illegal EcoRI site found at 4971
Illegal BglII site found at 4402
Illegal BglII site found at 5578 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4554
Illegal EcoRI site found at 4971
Illegal PstI site found at 5523 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4554
Illegal EcoRI site found at 4971
Illegal PstI site found at 5523
Illegal NgoMIV site found at 2622
Illegal NgoMIV site found at 2782
Illegal NgoMIV site found at 4370 - 1000COMPATIBLE WITH RFC[1000]
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