Difference between revisions of "Part:BBa K3352008"

Latest revision as of 12:34, 25 October 2020

T7 + New RBS & Terminator + SplintR Ligase Expressing Construct

The composite part utilizes a T7 Promoter (BBa_J64997), extended RBS (BBa_K3352002), SplintR Ligase (BBa_K3352000), and a T7 Terminator (BBa_K3352003).

Construct Design

We improved the constructs (BBa_K3352006) and (BBa_K33352007) by using pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. The pET vector includes the T7 promoter, which promotes high level transcription. By 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. These composite parts were synthesized by GenScript.

Figure 1: T7 + New RBS & Terminator + SplintR Ligase Expressing Construct

Characterization

pET3a and pET11a Promoter with T7 Terminator

We also aimed to improve this construct by using a pET3a and pET11a vectors with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 promoter, which promotes high level transcription [1]. By utilizing both a T7 promoter, T7 terminator, and an extended UTR sequence around the RBS and before the terminator, we can maximize protein expression for our enzymes (Figure 2) [1]. These composite parts were synthesized by GenScript.

Figure 2: Characterization of the pET T7 promoter with Φ29 polymerase (BBa_K3352009) construct and pET T7 promoter with SplintR (BBa_K3352008). We digested both constructs at the XbaI and PstI sites. The part (BBa_K3352001) was ligated into a pET11a backbone that is about 5500bps to form part (BBa_K3352009). Similarly, the part (BBa_K3352000) was ligated into pET3a backbone with a size of 4400bps which forms part (BBa_K3352008).

Protein Expression and Purification

We transformed our plasmids into BL21(DE3) E. coli cells. We grew bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The samples containing IPTG expressed the protein more strongly, which suggests that our protein was present (Figure 3).

Figure 3: SDS-PAGE results show that SplintR ligase and Φ29 polymerase was expressed by E. coli. We grew the bacterial cultures overnight at 37°C, diluted them to an OD600 of 0.2, and then grew them to 0.5, where we collected a sample of 1mL. We then added IPTG and grew the cultures for another 4 hours. After the additional 4 hours, we then collected another 1mL sample. We centrifuged both samples and resuspended the pellets in 1x Sample Buffer. The sample with the IPTG expressed the protein more strongly, which suggests that our protein was present at 68.2 kDa.

Figure 4: SDS-PAGE results show protein content at different steps of protein purification. A band around 35kDa was not present in the flow-through lane (red) or the wash buffer lanes, which corresponds with our expected His-tagged SplintR.

References

1. T7 Promoter System Vectors for Highest Expression Levels in Bacteria. (n.d.). Sigma-Aldrich. Retrieved October 22, 2020, from https://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/vector-systems/t7-promoter-system.html

Sequence and Features