Part:BBa_K4959001
SgaMcrA
Microorganisms use various defense systems to protect themselves against invading phages and mobile elements to ensure the stability of their genetic information, and nucleic acid endonucleases, which are capable of cleaving specific sequences or modifications, play an important role in this. Nucleases consist of a DNA recognition domain that binds to the target nucleic acid sequence and a cleavage domain that degrades the DNA.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 be targeted to cleave DNA with this modification. Based on the bioinformatics analyses, we have searched for potential sulfur-modification-dependent restriction enzymes.This part is one of the sulfur modified dependent restriction enzymes that we have screened.
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SgaMcrA (BBa_K4959001)
Profile
Name: SgaMcrA
Base Pairs: 1314 bp
Origin: Streptomyces gancidicus
Usage and Biology
Microorganisms use a variety of defense systems to protect themselves against invading phages and mobile elements to ensure the stability of their genetic information, and nucleic acid endonucleases, which are capable of cleaving specific sequences or specific modifications, play an important role in this. Nucleases consist of a DNA recognition domain that binds to the target nucleic acid sequence and a cleavage domain that degrades the DNA. DNA phosphorothionylation modifications (also known as sulfur modifications) are backbone modifications that replace non-bridging oxygen atoms on the phosphodiester bonds of DNA with sulfur atoms, and sulfur-modification-dependent restriction enzymes can target and cleave this type of modified DNA [1]. Bacterial defense systems overwhelmingly use nucleic acid endonucleases as weapons to degrade foreign DNA, of which, modification-dependent restriction enzymes are capable of limiting the invasion of modified DNA, and mainly include methylation modification-dependent restriction enzymes and sulfur modification-dependent restriction enzymes. Existing studies have found differences in the pH environments, cofactors, and concentrations used for the functioning of these classes of enzymes [2].
Construct Design
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.
As shown in Figure 5, non-pt DNA BL21 presents no significant sign of successful binding with normal DNA chain length – around 25 bp. Non-pt DNA BL21 presents various DNA chain lengths under the SDS PAGE test, including a normal chain with a length of around 25 bp and chains with a length of over 250 bp. The significantly longer chains observed on every DNA protein level (not including the control level) represent successful binding between the target enzyme and phosphorothioate B7A DNA.
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. Among them, Bis-Tris (pH6.0) and NaCl suitable pH and NaCl concentration for enzyme cleavage, MnCl2 provides the Mn2+ inducer while DTT maintains the oxidation state of Mn2+ cations.
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 6. 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.
[2] Yu H, Li J, Liu G, Zhao G, Wang Y, Hu W, Deng Z, Wu G, Gan J, Zhao YL, He X. DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses. Nucleic Acids Res. 2020 Sep 4;48(15):8755-8766.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 133
Illegal EcoRI site found at 550
Illegal PstI site found at 1102 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 133
Illegal EcoRI site found at 550
Illegal PstI site found at 1102 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 133
Illegal EcoRI site found at 550
Illegal BglII site found at 1157 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 133
Illegal EcoRI site found at 550
Illegal PstI site found at 1102 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 133
Illegal EcoRI site found at 550
Illegal PstI site found at 1102 - 1000COMPATIBLE WITH RFC[1000]
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