Difference between revisions of "Part:BBa K2918028"

Latest revision as of 06:24, 14 December 2019

WT T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator

Sequence and Features

The construct is 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.

Strain Construction

Aim: To clone the WT T7 promoter, Universal RBS, P5 and T7 terminator in a level 1 MoClo backbone pICH47761
Procedure: The DNA sequence of the part was cloned with the following Basic parts: BBa_K2918007, BBa_K2918014, BBa_K2918002 and BBa_K2918015. The cloning protocol can be found in the protocol section of our website!

Characterization of the SSB protein

For expressing our constructs 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 an 18% SDS-PAGE gel and mass spectrometry. From the 10 μL reaction, 8 μL was loaded on the SDS-PAGE while the other 2 μL was analysed by mass spectrometry.

SDS-PAGE
After expressing the SSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denature the protein the sample is treated with 2x SDS loading buffer with 10 mM dithiotreitol (DTT) for 10 minutes at 90°C. Samples were loaded on a 18% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Visualization was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488 nm laser and a band pass emission filter of 520 nm.

An SDS-PAGE was carried out for the p5 protein with three different promoter strengths (wild-type, medium and weak). To have a negative control, one expression reaction was run in the absence of DNA. In Figure 2, the three samples with different promoter strengths and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (13,3kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control.

Mass Spectrometry

Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. The mass spectrometer determines the mass of unique peptide sequences, and their elution time. For p5 these unique peptide sequences are: IFNAQTGGGQSFK and TVAEAASDLIDLVTR. 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 raw data and the optimized parameters for the mass spectrometry method can be found here and here.

The intensity of the mass spectrographs shown in Figure 3 only reflect the occurrence of a given sequence in the sample. These peptide sequences were only present in the samples that were expected. The difference in height can be attributed to the strength of the promoters, less peptides were measured with decreasing strength. For the first peptide IFNAQTGGGQSFK, the intensity of SSB with medium promoter and the weak promoter were 79% and 33% of the intensity of the WT promoter respectively. For the second peptide TVAEAASDLIDLVTR it is 80% and 37% respectively. In conclusion, the results were positive and the identity of the proteins could be further confirmed by mass spectrometry.

Toxicity

Our Sci-Phi 29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in E. coli BL21(DE3) pLysS. The results are shown in the graphs below.

The growth rate of p5 with a WT promoter is significantly lower than the growth rate of GFP. The trend is also observed upon IPTG induction (Figure 3). However, with a lower promoter strength, we see that the growth is actually better than that of GFP. This could be accounted to the fact that GFP actually also could be toxic as GFP produces H₂O₂. It is unclear why GFP toxicity is stronger than that of p5 with lower promoter strength. However, we can state that using a medium promoter the survival of the cells will increase.

If you want to know more, have a look at our Results page!

References

• 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.
• Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.