Part:BBa_K1354002:Experience
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Applications of BBa_K1354002
Terminal deoxynucleotidyl transferase (TdT) is one of the most promising DNA polymerases for de novo DNA synthesis because it can add nucleotides randomly to the initiation strand without a template. This year, our team LIUAN-Nanjing also tested various sources of Terminal deoxynucleotidyl transferase (TdT), including Bovine Terminal Deoxynucleotidyl Transferase (Part: BBa_K1354002), for recombinant expression activity in an E. coli expression system. We found that although Bovine Terminal Deoxynucleotidyl Transferase exhibits some catalytic activity, it is significantly lower compared to ZaTdT from Zonotrichia albicollis.
User Reviews
UNIQff3976fe3d747ff9-partinfo-00000000-QINU
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Cooper Union iGEM 2014 |
As seen from the polyacrylamide gel shown above, the TdT enzyme expressed in the Rosetta cells using our genetically engineered plasmid works as intended; base pairs are appended to oligos by the enzyme. However, there is a difference between the activity of our enzyme compared to the commercially obtained TdT. The majority of the oligos have only a few base pairs added by our expressed TdT while the lane of the commercial TdT is a smear of oligos, suggesting the commercial TdT has a greater reaction rate. But it can also be attributed to the fact that the 0.5 U/μL concentration of our expressed TdT is an overestimation. The TdT concentration of the reaction with our expressed TdT is less than that of the commercial TdT. There is also a large gap between the oligos with only a few bases added and those with 30+ bases added. This suggests that our expressed TdT has a higher processivity than that of commercially obtained TdT. This might be due to subtle sequence differences such as the His tag on our TdT sequence.
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LIUAN-Nanjing iGEM 2024 |
Terminal deoxynucleotidyl transferase (TdT) is one of the most promising DNA polymerases for de novo DNA synthesis because it can add nucleotides randomly to the initiation strand without a template. This year, our team LIUAN-Nanjing also tested various sources of Terminal deoxynucleotidyl transferase (TdT), including Bovine Terminal Deoxynucleotidyl Transferase (Part: BBa_K1354002), for recombinant expression activity in an E. coli expression system. We found that although Bovine Terminal Deoxynucleotidyl Transferase exhibits some catalytic activity, it is significantly lower compared to ZaTdT from Zonotrichia albicollis. |
