Difference between revisions of "Part:BBa K2918034"

Latest revision as of 12:59, 25 October 2020

Φ29 DNA polymerase (DNAP/p2)

DNA polymerase of the Φ29 bacteriophage.

Sequence and Features

The part has been confirmed by sequencing and there are no mutations.

Usage and Biology

The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, does not depend on specific sequences of DNA/RNA which simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2), terminal protein (TP/p3), single stranded binding protein (SSB/p5) and double stranded binding protein (DSB/p6). The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (OriL and OriR), which flank the protein-primed linear plasmid (Salas et al., 1994). The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases (Salas et al., 1994). The replication mechanism is depicted in Figure 1. If you want to read more about this mechanism, you can take a look at our Design page!

The Φ29 replication system is promising in many ways:

• The Φ29 DNA polymerase has the highest processivity of all known single subunit DNA polymerases (Blanco et al., 1989), and can be used for whole genome amplification.
• The Φ29 machinery along with cell-free expression systems can be used to establish the three dogmas of biology in vitro, setting the basis for artificial cell development.
• The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences.
• We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many new engineered protein-DNA complexes.
• Orthogonal replication enables not only replication independent from the host, but also the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes in vivo directed evolution a possibility.

Characterization

For expressing our phi29 DNAP we used PUREfrex 2.0. This is an E. coli based cell-free protein synthesis system and it contains all the elements to make in vitro translation-transcription possible. A 10 μL reaction consists of 5 μL feeding buffer, 0.5 μL enzyme solution, 1 μL ribosome solution, 5 nM DNA and RNAse-free milliQ for filling up the volume. For fluorescent labeling, 0.5 μL of BODIPY-Lys-tRNA_Lys (FluoroTectTM GreenLys, Promega) was added, this binds to the translation products at the lysine residues sites. The proteins were identified by mass spectrometry. From the 10-μL reaction, 2 μL was analyzed by the mass spectrometer.

Mass Spectrometer
Mass spectrometry was used to confirm the identity of the proteins. The mass spectrometer looks for the mass of unique peptide sequences, and their elution time. For DNAP these unique peptide sequences are: ENGALGFR and LVEGSPDDYTDIK. Data was normalized to the presence of the elongation factor EF-TU, which can be found in the same concentration in all PURE system reactions. The optimized parameters and raw data for the mass spectrometry method can be found here and here.

The intensity of the mass spectrographs shown in Figure 2 only reflect the occurrence of a given sequence in the sample. The successful expression of DNAP from our construct was confirmed by mass spectrometry as reported by Figure 1. In conclusion, the results were positive and the identity of the proteins could be further by mass spectrometry.

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone pICH41308 and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments (Weber et al, 2011). The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.

Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2.

Table 1: Overview of different level in MoClo

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.

Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Improvement

Improvement by TAS_Taipei 2020

Authors: Wilson Huang, Hannah Hsu

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 1). 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 2).

Figure 1: 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 2: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

• Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.
• Blanco, L., Lázaro, J. M., De Vega, M., Bonnin, A., & Salas, M. (1994). Terminal protein-primed DNA amplification.Proceedings of the National Academy of Sciences of the United States of America.
• Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. Plos ONE, 6(2), e16765.