Engineering

Transformation

For primer sequence please see Oligonucleotides

To produce Thermostable TdT (ThTdT), we designed a cloning strategy to express it in BL21 DE3 E. coli. This requires integrating the gene sequence of ThTdT into the expression plasmid pET-28b(+) plasmid.

P5+P6 primers were designed to inverse PCR amplify the pET-28b(+) plasmid to linearize and remove unwanted extra N- and C-terminus His6 regions. PCR will be performed using P9+P10 primers to amplify a 15bp overhang onto the ThTdT G-block (P11).

Therefore, those primers need to meet a few requirements:

1. complement with target plasmid
2. able to linearize target plasmid
3. contain overhang to allow downstream Gibson assembly

Primer detail please see Oligonucleotides

Following the last round of transformation in Cycle 1, we started to investigate possible reasons that may have caused this unsuccessful transformation. After consulting with iHP contact, Dr. Anthony Tang, we came up with possible improvements to our current design:

1. Increase Gibson assembly overhang regions from 15 base pairs (bp) to 30bp, P3+P4
2. Instead of using strain BL21 as transformation host, use DH5α or Top10 E. coli for increase transformation efficiency
3. Decrease the temperature for Gibson Assembly from 50ºC to 45ºC and run for 1h.
4. As a backup plan if all fails, try restriction enzyme digest to linearize the pET-28b vector, P12+P13

Taking advice from Dr. Tang, we decided to try out 30bp overhang primer (P3+P4) and DH5α E. coli transformation.

Following the transformation failure from the previous design cycle, we used DH5α and Top10 E. coli provided by the Hallam Lab to perform the transformation of the 15bp and 30bp overhang Gibson assembly products.

Based on the consistent failure in transformation from previous cycles, we decided to perform restriction digest cloning using XhoI and NcoI restriction enzyme. The restriction enzymes will linearized pET-28b(+) plasmid. The ThTdT G-block will be PCR amplified with the restriction enzyme recognition site followed by gibson assembly ligation of pET-28b(+) plasmid and ThTdT G-block.

Based on the consistent failure in transformation throughout all previous design cycles, we decided to perform transformation using DH5α chemically competent E. coli cells from the Sorensen Lab in this cycle.