Difference between revisions of "Part:BBa K4623013"
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==Usage and Biology== | ==Usage and Biology== | ||
| − | The Nucleotide Silinker (NS) is a novel engineered protein that covalently links nucleic acid ends to biotinylated adapters. The sequence in FASTA format includes an added HIS tag, enabling purification of the NS protein using a nickel column. Upstream of the mSA sequence, a TrxA (BBa_K3619001) fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site, allowing binding to biotinylated functional proteins. The TrwC protein can covalently link specific 5' single-stranded nucleotide sequences, facilitating the conversion between nucleic acids and protein connectors. | + | The Nucleotide Silinker (NS) is a novel engineered protein that covalently links nucleic acid ends to biotinylated adapters. The sequence in FASTA format includes an added HIS tag, enabling purification of the NS protein using a nickel column. Upstream of the mSA sequence, a TrxA (BBa_K3619001) fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site, allowing binding to biotinylated functional proteins. The TrwC (BBa_K4623003) protein can covalently link specific 5' single-stranded nucleotide sequences, facilitating the conversion between nucleic acids and protein connectors. |
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===Induction Condition=== | ===Induction Condition=== | ||
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| − | After introducing the pETDuet1-NS plasmid into our engineered BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited very low expression levels under IPTG induction (as seen in the figure below), with low cell density. Therefore, in subsequent experiments, we employed the BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. However, after transformation, no visible colonies were observed. It is suspected that the NS protein may have significant toxicity to the engineered bacteria and is incompatible with the pLysS plasmid. | + | |
| + | After introducing the pETDuet1-NS plasmid into our engineered <i>E.coli</i> BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited very low expression levels under IPTG induction (as seen in the figure below), with low cell density. Therefore, in subsequent experiments, we employed the <i>E.coli</i> BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. However, after transformation, no visible colonies were observed. It is suspected that the NS protein may have significant toxicity to the engineered bacteria and is incompatible with the pLysS plasmid. | ||
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Latest revision as of 13:45, 11 October 2023
Nucleotide Silinker(NS)
Usage and Biology
The Nucleotide Silinker (NS) is a novel engineered protein that covalently links nucleic acid ends to biotinylated adapters. The sequence in FASTA format includes an added HIS tag, enabling purification of the NS protein using a nickel column. Upstream of the mSA sequence, a TrxA (BBa_K3619001) fusion tag is added to aid protein folding and reduce the formation of inclusion bodies in bacterial cells. Following protein expression, cleavage by thrombin exposes the mSA (BBa_K4623001) site, allowing binding to biotinylated functional proteins. The TrwC (BBa_K4623003) protein can covalently link specific 5' single-stranded nucleotide sequences, facilitating the conversion between nucleic acids and protein connectors.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1024
Illegal PstI site found at 1342 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1024
Illegal PstI site found at 1342 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1024
Illegal PstI site found at 1342 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1024
Illegal PstI site found at 1342
Illegal AgeI site found at 448
Illegal AgeI site found at 508 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1147
Illegal SapI.rc site found at 862
Cultivation, Purification and SDS-PAGE
Induction Condition
After introducing the pETDuet1-NS plasmid into our engineered E.coli BL21(DE3) bacteria, we conducted a small-scale trial expression and found that BL21(DE3) exhibited very low expression levels under IPTG induction (as seen in the figure below), with low cell density. Therefore, in subsequent experiments, we employed the E.coli BL21(DE3) pLysS strain, which carries the pLysS plasmid and expresses T7 lysozyme to suppress leaky expression caused by T7 RNA polymerase. However, after transformation, no visible colonies were observed. It is suspected that the NS protein may have significant toxicity to the engineered bacteria and is incompatible with the pLysS plasmid.
