Part:BBa_K3352007

T7 + RBS Phi29 DNA Polymerase Expressing Construct

The composite part utilizes a T7 Promoter (BBa_J64997), a ribosome binding site (BBa_B0034), Φ29 polymerase (BBa_K3352001), and a double terminator (BBa_K0015).

Construct Design

We attached a 6x His-tag upstream of the Φ29 DNA polymerase for purification purposes followed by a GS linker to allow flexibility between tag and Φ29. We then flanked the open reading frame with upstream T7 promoter and strong ribosome binding site (RBS) (BBa_B0034) and downstream double terminator (BBa_B0015). This entire composite part was gene synthesized by IDT.

Figure 1: Φ29 DNA polymerase with T7 Promoter, RBS and Double Terminator Construct

Characterization

T7 Promoter and Strong RBS

Seeing that purified Φ29 DNA polymerase is fundamental to the development of our diagnostic test, we attempted to resolve the issue of low protein expression by replacing the strong promoter in our constructs with a T7 promoter and expressing our protein in BL21(DE3) E. coli [2]. BL21(DE3) strains contain the chromosomal gene T7 RNA polymerase, which is regulated by a lac promoter [1]. T7 RNA polymerase has been found to be highly selective and efficient in transcribing only the T7 promoter [1, 2]. Resulting in almost a five-fold faster elongation rate than E. coli RNA polymerase, T7 would be a much stronger promoter of choice. Thus, by using IPTG during protein expression to activate the lac promoter, and thus the T7 RNA polymerase of our BL21(DE3) E. coli culture, we can significantly increase the production of our enzymes positioned downstream of our T7 promoter [1 ,2]. We obtained the sequence of the T7 promoter (BBa_J65997) from the Parts Registry and used it to replace the strong promoters on our Φ29 DNA polymerase construct. This part was synthesized by Twist Biosciences and IDT.

Protein Expression and Purification

We transformed our newly designed plasmids into BL21(DE3) E. coli cells. We grew overnight cultures and then diluted and grew cells to OD600 0.5. We then induced expression with 0.1 M IPTG and allowed cultures to grow an additional 2 hours. We harvested cells and then lysed them with xTractor Lysis Buffer [3]. We purified our His-tagged proteins using Ni sepharose affinity chromatography. In order to check if our proteins were correct, we used SDS-PAGE. Our results showed Φ29 DNA polymerase migrated at the expected sizes of 68.2 kDa.

Figure 2: Our SDS-PAGE results show that E. coli is able to produce Φ29 DNA polymerase. We grew bacterial cultures overnight at 37°C. We then lysed and prepared samples for SDS-PAGE. The expected size is listed on the side.

Figure 3: In our improved construct, we induced the T7 Promoter and the SDS-PAGE results showed that our band was expressed strongly.

References

1. Biolabs, N. E. (n.d.-a). E. coli Expression Strains | NEB. Retrieved October 22, 2020, from https://international.neb.com/products/competent-cells/e-coli-expression-strains/e-coli-expression-strains

2. Arnaud-Barbe, N. (1998). Transcription of RNA templates by T7 RNA polymerase. Nucleic Acids Research, 26(15), 3550–3554. https://doi.org/10.1093/nar/26.15.3550

3. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 640
  Illegal SapI.rc site found at 1038