Difference between revisions of "Part:BBa K3352004"

Latest revision as of 03:07, 26 October 2020

Strong Promoter and RBS SplintR Ligase Expressing Construct

The composite part utilizes a strong promoter (BBa_J23100), a ribosome binding site, SplintR ligase(BBa_K3352000), and a double terminator (BBa_K0015).

Figure 1: SplintR ligase with strong promoter, RBS, and double terminator

Construct Design

We optimized the DNA sequence for expression in E. coli and removed the PstI cutting site. We attached a 6x histidine tag (6x His-Tag) upstream of the SplintR ligase sequence for purification purposes followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3352000). We flanked the open reading frame with an 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.

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 was also around 2.7kB. BBa_K3352006 was obtained from Twist Bioscience and was cloned into the ampicillin backbone (pSB1A3).

Characterization

Protein Expression and Purification

We transformed our designed plasmids (BBa_K3352004) into DH5⍺ E. coli cells. We grew overnight cultures, diluted those cultures, and then 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 construct 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 35 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.-c). SplintR® Ligase | NEB. Retrieved October 20, 2020, from https://international.neb.com/products/m0375-splintr-ligase

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

Sequence and Features