Overview
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 cleavage of this type of modified DNA. This project searched for three potential sulfur modification-dependent restriction enzymes based on bioinformatics analysis: sga,sva, and asp.

 

While the COVID-19 pandemic in previous years greatly stimulated the nucleic acid detection industry in China, the patent of popular techniques such as those based on the CRISPR-Cas9, however, remained abroad. This greatly increased the cost of production and decreased the freedom of application with such technologies. In response, our team delved into the binding and cleavage mechanisms of restriction endonucleases that target phosphorothioated DNA. During our research, we encountered information not readily available in existing parts registry. Consequently, we have embarked on adding new parts to the database to better represent our findings and facilitate future research.

 

Table 1. Parts Collection

Part number	Part name	Part type	Part property	Contribution Type
BBa_K3521004	pet28a-backbone	composite part	vector	New experimental data
BBa_K4959000	AspMcrA	basic part	Coding	New part
BBa_K4959001	SgaMcrA	basic part	Coding	New part
BBa_K4959002	SvaMcrA	basic part	Coding	New part
BBa_K4959003	AspMcrA-pET28a	composite part	Plasmid	New part
BBa_K4959004	SgaMcrA-pET28a	composite part	Plasmid	New part
BBa_K4959005	SvaMcrA-pET28a	composite part	Plasmid	New part

 

Create a New Basic Part ：  BBa_K4959000, AspMcrA

BBa_K4959000 is a coding sequence of Sulfur-modification-dependent restriction enzymes AspMcrA. Microorganisms employ a diverse array of defense systems to shield their genetic information from the intrusion of phages and other mobile genetic elements. Central to these defenses are nucleic acid endonucleases, which possess the ability to recognize and cleave specific DNA sequences or modifications, thereby playing a pivotal role in safeguarding the microorganism's genetic integrity.

 

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 and investigated their functional domains accordingly.

 

 

Create a New Basic Part ：  BBa_K4959001, SgaMcrA

BBa_K4959001 is a coding sequence of Sulfur-modification-dependent restriction enzymes SgaMcrA. Based on the bioinformatics analyses, we have searched for potential sulfur-modification-dependent restriction enzymes and investigated their functional domains accordingly.

Create a New Basic Part ：  BBa_K4959002, SvaMcrA

BBa_K4959002 is a coding sequence of Sulfur-modification-dependent restriction enzymes SvaMcrA. Based on the bioinformatics analyses, we have searched for potential sulfur-modification-dependent restriction enzymes and investigated their functional domains accordingly.

Create a New Composite Part: BBa_K4959003, AspMcrA-pET28a.

AspMcrA-pET28a is a novel plasmid constructed using the pET-28a vector and a gene fragment ca lled AspMcrA. Introduction of this recombinant plasmid into BL21 competent cell produces our target  protein, a sulfur-modification-dependent restriction enzyme. The protein enables us to conduct the next functional test  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. Profile of pET28a-asp

Create a New Composite 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 our target protein, , a sulfur-modification-dependent restriction enzyme. The protein produced enables us to conduct the next functional test  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 2. Profile of pET28a-sga

 

Create a New Composite Part: BBa_K4959005, SvaMcrA-pET28a.

SvaMcrA -pET28a is a novel plasmid constructed using the pET-28a vector and a gene fragment called SvaMcrA. 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 test  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 3. Profile of pET28a-sva