Part:BBa_K4959003

AspMcrA-pET28a

AspMcrA-pET28a is a novel plasmid constructed using the pET-28a vector and a gene fragment called AspMcrA. 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.

AspMcrA-pET28a (BBa_K4959003)

Construction Design

AspMcrA-pET28a is a novel plasmid constructed using the pET-28a vector (BBa_K3521004) and a gene fragment called AspMcrA (BBa_K4959000). 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.

Figure 1. Gel electrophoresis results of vectors and target gene fragments

Engineering Principle

DNA phosphorothionylation modification (also known as sulfur modification) is a backbone modification that replaces non-bridging oxygen atoms on the phosphodiester bond of DNA with sulfur atoms, and sulfur modification-dependent restriction enzymes can target and cleave this type of modified DNA[1]. sulfur modification-dependent restriction enzymes bind strongly to nucleic acids, and the investigation of the binding and cleavage mechanisms will help to apply them as important components in thermostable nucleic acid detection. In this project, we plan to synthesize the potential sulfur-modification-dependent restriction enzyme, purify the protein by constructing it in an expression vector, and investigate the binding and cleavage activities of the protein by using EMSA assay and cleavage assay.

Figure 2. Engineering Principle

Cultivation, Purification, and SDS-PAGE

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.

Figure 3. Gel electrophoresis results of vectors and target gene fragments

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.

Figure 4. Growth of recombinant plasmids of Sga, Sva, and Asp after transformation of Escherichia coli in plate culture

In the second phase of the experiment, we transformed the recombinant plasmid into BL21 E. coli receptor cells and allowed them to grow in liquid LB medium. When the bacteria grew, we induced expression of the target proteins using IPTG inducers. After overnight induction, we extracted the target proteins by told centrifugation and ultrasonic fragmentation and performed protein purification.

Figure 5. SDS PAGE results of the target protein

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 Asp 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.

Figure 6. Result of BL21 and B7A EMSA for Asp

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). We expect to see cleavage only on ptDNA while no successful cleavage on non-ptDNA.

Figure 7. AGE result of BL21 & B7A Nucleic Acid Cleavage

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 244
  Illegal PstI site found at 457
  Illegal PstI site found at 904
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 244
  Illegal PstI site found at 457
  Illegal PstI site found at 904
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 143
  Illegal BglII site found at 5341
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 244
  Illegal PstI site found at 457
  Illegal PstI site found at 904
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 244
  Illegal PstI site found at 457
  Illegal PstI site found at 904
  Illegal NgoMIV site found at 3561
  Illegal NgoMIV site found at 3721
  Illegal NgoMIV site found at 5309
  
• 1000
  COMPATIBLE WITH RFC[1000]