Difference between revisions of "Part:BBa K3352000"

Revision as of 06:59, 20 October 2020

SplintR Ligase with His-Tag and GS Linker Sequence

SplintR Ligase catalyzes the ligation of adjacent single-stranded DNA splinted by complementary RNA strands. SplintR Ligase has been previously shown to be capable of differentiating these ligation junctions to SNP levels and ligate padlock probes.

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 ORF.

PCR Results

Insert gel pic

Characterization

Strong Promoter and Strong RBS

We flanked the open reading frame with upstream strong promoter and strong ribosome binding site (RBS) combination (BBa_K880005) and downstream double terminator (BBa_B0015).

Protein Expression and Purification

We prepared overnight DH5⍺ E. coli cultures and obtained OD600 readings to dilute cultures to standardized populations. We grew the cultures to log phase, and lysed cells with xTractor Lysis Buffer (Takara Bio). We purified our His-tagged proteins using Ni sepharose affinity chromatography, then ran our prepared samples through a nickel column in order to purify out our his-tagged proteins. In order to check if our proteins were correct, we used a SDS-PAGE protein gel electrophoresis. However, our constructs that used a strong promoter and strong RBS combination yielded proteins were not expressed.

Improved Design

T7 Promoter and Strong RBS

Seeing that purified Φ29 DNA Polymerase and SplintR Ligase are fundamental to the development of our diagnostic test, we attempted to resolve the issue by introducing a T7 promoter to our construct and expressing our protein with BL21 (DE3) E. coli (Figure 6-7). DE3 strains contain the chromosomal gene T7 RNA Polymerase which is regulated by a lac promoter. T7 RNA Polymerase has been found to be highly selective and efficient in transcribing only the T7 promoter. Resulting in almost a five-fold faster elongation rate that E. coli RNA Polymerase, T7 would be a much stronger promoter of choice. Thus, by using IPTG during protein synthesis of our BL21 (DE3) E. coli culture, we would effectively produce more T7 RNA Polymerase and significantly increase the production of our enzymes positioned downstream of our T7 promoter.

Protein Expression and Purification

Our SDS-PAGE results show that E. coli is able to produce SplintR Ligase. Bacterial cultures were grown overnight at 37°C, lysed, and prepped for SDS-PAGE. The expected size is listed on the side.

pET3a T7 Promoter

We also aimed to improve this construct by using pET3a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 bacteriophage gene 10, which promotes high level transcription and translation. Utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we would maximize the protein expression for our enzymes.

Protein Expression and Purification

Our SDS-PAGE results show that both purified proteins migrate at the expected sizes. However, due to the presence of unfavorable buffer conditions in our eluted proteins, we performed a buffer exchange to reach the desired pH and storage conditions for our enzymes. We used these proteins for our viral detection test.

Sequence and Features