Part:BBa_K2918028
WT T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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.
Overview
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 (Nies et al., 2018). 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 (Nies et al., 2018).
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-tRNALys (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 the mass spectrometer.
SDS-PAGE
After expressing the SSB 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.
An SDS-PAGE was carried out for the SSB protein with 3 different promoter strengths: Wild-Type, 0.5 and 0.1. For a control PURE solution without any DNA was used. As can be concluded from the figure, in the sample containing the p5 protein a band indicated by the asterix can be found at the expected molecular weight(13kb). The band is also absent in the control, indicating that the p5 protein was successfully produced in the PURE system using this construct. The other band that can also be seen in the control could be due to contamination
Mass Spectrometry
Next to the SDS-PAGE, 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 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.
The intensity of the mass spectrographs shown in Figure 2 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 0.5 promoter and the 0.1 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.
In Vitro replication
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.
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