Difference between revisions of "Part:BBa K3352001"

Latest revision as of 03:11, 26 October 2020

Φ29 DNA Polymerase with His-Tag and GS linker Sequence

Φ29 DNA polymerase synthesizes new strands of DNA with strand displacement for any existing DNA strands in front [1]. It also has exonuclease activity from the 3’ to 5’ direction, usually only responsible for the cutting of single-stranded nucleic acids but has been shown to be capable of cutting complementary strands as well [1].

Construct Design

We attached a 6x His-tag upstream of the Φ29 DNA polymerase for purification purposes followed by a GS linker to allow flexibility between tag and Φ29. We then flanked the open reading frame with 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.

Figure 1: Φ29 DNA polymerase with His-Tag and GS linker

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

Characterization

Protein Expression and Purification of Φ29 DNA polymerase

We transformed our designed plasmids (BBa_K3352005) into DH5⍺ E. coli cells. We grew overnight cultures, diluted those cultures, and 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 and Φ29 polymerase constructs 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 68 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.

Improved Design

T7 Promoter and Strong RBS

Seeing that purified Φ29 DNA polymerase is fundamental to the development of our diagnostic test, we attempted to resolve the issue of low protein expression by replacing the strong promoter in our constructs with a T7 promoter and expressing our protein in BL21(DE3) E. coli [4]. BL21(DE3) strains contain the chromosomal gene T7 RNA polymerase, which is regulated by a lac promoter [3]. T7 RNA polymerase has been found to be highly selective and efficient in transcribing only the T7 promoter [3, 4]. Resulting in almost a five-fold faster elongation rate than E. coli RNA polymerase, T7 would be a much stronger promoter of choice. Thus, by using IPTG during protein expression to activate the lac promoter, and thus the T7 RNA polymerase of our BL21(DE3) E. coli culture, we can significantly increase the production of our enzymes positioned downstream of our T7 promoter [3,4]. We obtained the sequence of the T7 promoter (BBa_J65997) from the Parts Registry and used it to replace the strong promoters on our Φ29 DNA polymerase construct. This part was synthesized by Twist Biosciences and IDT.

Protein Expression and Purification

We transformed our newly designed plasmids into BL21(DE3) E. coli cells. We grew overnight cultures and then diluted and grew cells to OD600 0.5. We then induced expression with 0.1 M IPTG and allowed cultures to grow an additional 2 hours. We harvested cells and then lysed them with xTractor Lysis Buffer [6]. We purified our His-tagged proteins using Ni sepharose affinity chromatography. In order to check if our proteins were correct, we used SDS-PAGE. Our results showed Φ29 DNA polymerase migrated at the expected sizes of 68.2 kDa.

Figure 4: Our SDS-PAGE results show that E. coli is able to produce Φ29 DNA polymerase. We grew bacterial cultures overnight at 37°C. We then lysed and prepared samples for SDS-PAGE. The expected size is listed on the side.

Figure 5: In our improved construct, we induced the T7 Promoter and the SDS-PAGE results showed that our band was expressed strongly.

pET11a T7 Promoter

We also aimed to improve this construct by using a pET11a vector with appropriate BioBrick prefixes and suffixes that fulfill the assembly standard. pET vectors include the T7 promoter, which promotes high level transcription [5]. 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 6)[5]. These composite parts were synthesized by GenScript.

Figure 6: 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 7).

Figure 7: Figure 7: SDS-PAGE results show that 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 1mLd. 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.

Improvement of an Existing Part

We optimized the protein coding region for Φ29 DNA polymerase for high expression in E. coli. We also inserted 3 additional codons that should be present as found in the Φ29 DNA polymerase amino acid sequence in the parent organism Bacillus phage Φ29. To isolate the enzyme after expression, we added a N-terminal 6x histidine tag to the Φ29 DNA polymerase sequence through a GS linker (BBa_K3352001). This improves the existing Φ29 DNA polymerase sequence (BBa_K2918034) from team TUDelft 2019. To test whether our improved sequence was better, we expressed our Φ29 DNA polymerase (BBa_K3352009), which contains (BBa_K3352001) alongside TUDelft’s (BBa_K2918034) and analyzed the expression levels by SDS-PAGE. We grew bacterial cultures overnight at 37°C. We then diluted the cultures to an OD600 of 0.2 and grew them to an OD600 of 0.5, at which point we collected a 1mL sample. We then added IPTG and grew the cultures for another 4 hours after which another 1mL sample was collected. We centrifuged all samples and resuspended the pellets in 1x Sample Buffer and performed SDS-PAGE analysis. In our protein gel, we saw that our construct was able to better express Φ29 polymerase, especially after inducing it with IPTG, relative to the pre-existing part (Figure 8). To further confirm that the observed band was indeed Φ29 polymerase, we purified the enzyme using Ni sepharose affinity chromatography. The results showed a clear band at 68.2 kDa for the elution fractions that were not present in the flow-through or wash fractions, indicating that we successfully expressed and purified His-tagged Φ29 polymerase (Figure 9).

Figure 8: Comparison between part (BBa_K2918034) and part (BBa_K3352009) showing our improved Φ29 construct. We can clearly see a band that is around 68kDa in the post-induced pET T7 promoter and Φ29 construct (red) that is not present in the post-induced TUDelft 2019 Φ29 construct (blue), which suggests that our construct can better express Φ29 DNA polymerase.

Figure 9:SDS-PAGE results show protein content at different steps of protein purification. A band around 68kDa was not present in the flow-through lane (red) or the wash buffer lanes, which corresponds with our expected His-tagged Φ29.

References

1. Biolabs, N. E. (n.d.-b). Phi29 DNA Polymerase | NEB. Retrieved October 20, 2020, from https://international.neb.com/products/m0269-phi29-dna-polymerase

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

3. Biolabs, N. E. (n.d.-a). E. coli Expression Strains | NEB. Retrieved October 22, 2020, from https://international.neb.com/products/competent-cells/e-coli-expression-strains/e-coli-expression-strains

4. Arnaud-Barbe, N. (1998). Transcription of RNA templates by T7 RNA polymerase. Nucleic Acids Research, 26(15), 3550–3554. https://doi.org/10.1093/nar/26.15.3550

5. 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