Part:BBa_K3352000
SplintR Ligase with His-Tag and GS Linker Sequence
SplintR Ligase catalyzes the ligation of adjacent single-stranded DNA splinted by ole 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
Figure #: This is a DNA Gel electrophoresis for parts BBa_K3352004, BBa_K3352005, BBa_K3352006 and BBa_K3352007, which shows the phi29 and SplintR plasmids. All four constructs were ordered from Twist or IDT and all conform to a biobrick assembly standard 10 and were digested with Ecor1 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). <b/>
<b> Characterization
Strong Promoter and Strong RBS <b> 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).
<b> Protein Expression and Purification <b> 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.
<b> Figure #: SDS-PAGE results show that E. coli carrying BBa_K3352004 produce SplintR Ligase. The expected size for SplintR Ligase is 35.7 kDa, though we observed a band, it is not expressed strongly.
Improved Design
T7 Promoter and Strong RBS <b> 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.
<b> Protein Expression and Purification <b>
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.
<b> pET3a T7 Promoter <b> 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.
<b> Protein Expression and Purification <b>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 790
- 1000COMPATIBLE WITH RFC[1000]
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