Difference between revisions of "Part:BBa K2918023"

Revision as of 08:01, 21 October 2019

Weak 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

The construct is confirmed by sequencing and there are no mutations.

Overview

The replication of DNA and its conversion into functional proteins are vital processes in all living systems. DNA is copied during the replication process. The bacteriophage Φ29 contains a DNA replication machinery which replicates the linear plasmid by itself. This process is called orthogonal replication and can be beneficially used. The desired gene can be expressed in other hosts without interfering with the genome of its host. Our Sci-Phi 29 tool is based on the Φ29 DNA replication system and its four proteins. The terminal proteins (TP) cap the linear DNA, protect the linear DNA and are the primer for initiation of replication by the Φ29 DNA polymerase (DNAP/p2). DNAP binds to the TP and replicates the DNA. During the replication, single and double stranded DNA is protected by respectively single stranded DNA binding proteins (SSB/p5) and double stranded DNA binding proteins (DSB/p6).

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_K2918005, 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 analysed 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.

An SDS-PAGE was carried out for the DSB protein with 3 different promoter strengths: Wild-Type, Medium and Weak. 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(12kDa). 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 that can also 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 looks for 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.

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 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.