We improved the characterization of BBa_J64997. The part T7 consensus -10 and rest serves as a T7 promoter that allows the binding of T7 RNA Polymerase to initiate transcription (Arnaud-Barbe, 1998). We decided to further characterize this part to provide data on whether this promoter sequence serves a benefit to the expression of enzymes compared to other promoters, specifically enzymes such as ligases, which are not only indispensable in bioanalytical techniques but also in viral diagnostic kits such as ours.
In order to characterize this existing part, we flanked a pET3a T7 promoter with downstream BBa_K3352002, BBa_K3352000, and BBa_K3352003, which forms the composite part BBa_K3352008. Since the pET3a T7 promoter has the same sequence as the T7 consensus -10 and rest, the SplintR Ligase enzyme expressed through this construct serves as an indicator of protein expression levels for this T7 promoter sequence.
Figure 1: Design of pET Parts + SplintR Ligase Expressing Construct (BBa_K3352008).
Plasmids containing this construct was transformed into DH5⍺ E. coli cells for plasmid replication and subsequently miniprepped to obtain a high yield of plasmid. We then transformed these plasmids into BL21(DE3) E. coli cells. Growing an overnight culture and measuring the OD600 to dilute cells to standardized populations, we induced expression with 0.1M IPTG once the OD600 surpassed 0.5. Liquid cultures were grown for an additional 2 hours.
Figure 2: SDS-PAGE on protein expression before and after IPTG induction with the pET3a Parts SplintR Ligase expressing construct (BBa_K3352008).
While growing our cultures, we collected culture samples both before and after IPTG induction. We subsequently ran SDS-PAGE with these samples. Our results in Figure 2 indicates that bands of the enzyme were in fact present at the 35.2kDa band, the molecular weight of our SplintR Ligase enzyme (including the 6x histidine tag and GS linker attached). However, due to the presence of other notable proteins, we seeked further validation to ensure that our enzyme of interest was expressed.
After protein expression, we harvested and lysed the cells with xTractor Lysis Buffer and centrifuged them for sample preparation (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.). This was followed by protein purification through Ni sepharose affinity chromatography, which could isolate our his-tagged SplintR Ligase enzymes.
Figure 3: SDS-PAGE on purified proteins with the pET3a Parts SplintR Ligase expressing construct (BBa_K3352008).
We once again ran SDS-PAGE but with the purified samples. These results shown in Figure #3 once again suggests that our now purified enzyme of interest, SplintR Ligase, was produced through the expression of our construct design as seen through the band at 35.2kDa.
To compare the protein expression capability of this T7 promoter, we conducted the same test but replaced our T7 promoter from our previously used composite part BBa_K3352008 with a strong constitutive promoter BBa_J23100. This formed our construct BBa_K3352004, whose SplintR Ligase expression can be compared to that of BBa_K3352008.
Figure 4: Design Strong Promoter SplintR Ligase Expressing Construct (BBa_K3352004).
Due to the usage of a promoter that isn’t T7, we used both DH5⍺ E. coli cells for both plasmid replication and protein expression (Arnaud-Barbe, 1998; Biolabs, n.d.; T7 Promoter System Vectors for Highest Expression Levels in Bacteria, n.d.). IPTG also served no purpose in inducing protein expression as T7 RNA Polymerase is not relied on (Biolabs, n.d.). Beside these alterations, the transformation, culturing, protein expression and purification were conducted in the same manner.
Figure 5: SDS-PAGE on purified proteins with the strong promoter SplintR Ligase expressing construct (BBa_K3352004) and strong promoter phi29 DNA Polymerase expressing construct (BBa_K3352005).
The SDS-PAGE results for the purified SplintR Ligase expressed through the strong promoter showed an array of enzymes and a faint band at the 35.22kDa lane. Upon further investigation, the presence of a similar band on the purified phi29 DNA Polymerase expressed through the strong promoter further shows ambiguity in our results, highly indicating that the protein band for the purified SplintR Ligase may not be SplintR Ligase at all.
This provides convincing evidence that the T7 promoter expresses certain proteins that cannot be properly expressed through other promoters. In this case, the protein tested would be SplintR Ligase, and could suggest that the expression of these crucial enzymes for viral testing should be conducted through a T7 construct.