Part:BBa_K2918025

WT T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator

This part consists of a T7 promotor, a universal Ribosome Binding Site (RBS), a Coding DNA Sequence (CDS) coding for the DSB p6 and a Wild Type T7 terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 292
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 68
  Illegal BsaI.rc site found at 94
  

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, do not depend on specific sequences of DNA/RNA and 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 (DSB/p6) 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!

• 

  Figure 1: Overview of phi 29 replication mechanism

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., 1988), 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 not only enables replication independent from the host, but the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome makes in vivo directed evolution a possibility.

Strain Construction

Aim: To clone the promoter in a level 1 MoClo backbone pICH47751
Procedure: The DNA sequence of the part was cloned with the following Basic parts: BBa_K2918007, BBa_K2918014, BBa_K2918003 and BBa_K2918015. The cloning protocol can be found in the protocol section of our website!

Characterization of the DSB 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 analyzed by the mass spectrometer.

SDS-PAGE
After expressing the DSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denaturate the protein the sample is also 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.

• 

  Figure 2: SDS-PAGE gels of DSB after protein purification.

An SDS-PAGE was carried out for the DSB protein with three different promoter strengths: Wild-Type, Medium and Weak. PURE solution without any DNA was used as control. As can be concluded from the figure, in the sample containing the p6 protein a band indicated by the asterix can be found at the expected molecular weight (12 kDa). The band is also absent in the control, indicating that the p6 protein was successfully produced in the PURE system using this construct. The other band which can be seen in the control could be due to contamination of the milliQ or the master mix containing the Purefrex solutions.

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 p6 these unique peptide sequences are: GEPVQVVSVEPNTEVYELPVEK and FLEVATVR. 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.

• 

  Figure 3A: Identification in mass spectrometry of one peptide (FLEVATVR) of p6 sample after purification
• 

  Figure 3B: Identification in mass spectrometry of one peptide (GEPVQVVSVEPNTEVYELPVEK) of p6 sample after purification

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 GEPVQVVSVEPNTEVYELPVEK, the intensity of DSB with Medium promoter and the Weak promoter were 57% and 35% of the intensity of the WT promoter respectively. For the second peptide FLEVATVR it is 82% and 35% 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.

• 

  Figure 3A: Normalized maximum growth rate of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction
• 

  Figure 3B: Normalized maximum growth rate of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction
• 

  Figure 3C: Normalized maximum growth rate of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction

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.