Part:BBa_K3352005

Strong Promoter and RBS phi29 DNA Polymerase Ligase Expressing Construct

The composite part utilizes a strong promoter (BBa_J23100), a ribosome binding site, Φ29 polymerase (BBa_K3352001), and a double terminator (BBa_B0015).

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 strong promoter and strong ribosome binding site (RBS) combination (BBa_K880005) and downstream double terminator (BBa_B0015). This entire composite part was gene synthesized by IDT.

Figure 1: Φ29 DNA polymerase with His-Tag and GS linker

Results

Figure 2: Characterization of our Φ29 polymerase, parts BBa_K3352004 and BBa_K3352005, and SplintR ligase, BBa_K3352006 and BBa_K3352007. All four constructs were ordered from Twist or IDT, conformed to a biobrick assembly standard 10, and digested with EcoRI and PstI. Parts BBa_K3352004 and BBa_K3352005 were ordered from IDT and had a kanamycin backbone (pUCIDT KAN), which had a size of 2.7kB. BBa_K3352007 was also ordered from IDT, however, it contained an ampicillin backbone (pUCIDT AMP), which is also around 2.7kB. BBa_K3352006 was obtained from Twist Bioscience and was cloned into the ampicillin backbone (pSB1A3).

Characterization

Protein Expression and Purification of Φ29 DNA polymerase

We transformed our designed plasmids (BBa_K3352005) into DH5⍺ E. coli cells. We grew overnight cultures, diluted those cultures, and grew the cells to log phase. We lysed cells with xTractor Lysis Buffer (Takara Bio) and purified our His-tagged proteins using Ni sepharose affinity chromatography [2]. In order to check if our proteins were correct, we used SDS-PAGE.

Based on our results, our SplintR ligase and Φ29 polymerase constructs that used a strong promoter and strong RBS combination (BBa_K3352004 and BBa_K3352005) did not express an appreciable amount of protein (Figure 3).

Figure 3: SDS-PAGE results show protein content at different steps of protein purification. A band around 68 kDa in the cell lysate (blue) and the eluate (red), matches our expected His-tagged Φ29. However, many other proteins were present in the eluate and in the flowthrough lane (yellow). This prompted us to redesign our constructs.

References

1. Biolabs, N. E. (n.d.-b). Phi29 DNA Polymerase | NEB. Retrieved October 20, 2020, from https://international.neb.com/products/m0269-phi29-dna-polymerase

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 656
  Illegal SapI.rc site found at 1054