Difference between revisions of "Part:BBa K2918023"

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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

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!

Identification of the DSB protein

For identifying our constructs we used PURE(Protein synthesis Using Recombinant Elements) system. 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 0.5 μL enzym solution, 1 μL ribosome solution, 0.5 μL Green Lyse, 5 nM DNA and RNAse-free milliQ for filling up the volume. 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 reserved for the mass spectrometer.

SDS-PAGE

Figure

An SDS-PAGE was carried out for the DSB 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 p6 protein a band can be found at the expected height(13kb). The band is also absent in the control, indicating that the p6 protein was successfully produced in the PURE system using this construct.

Mass Spectrometry

Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. To do this, a sequence unique to the DSB’s amino acid sequence were chosen and screened for their presence in the PURE system. For the p6 the 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 amount in all PURE system solutions.

The intensity of the mass spectrographs shown in Figure 2 only reflect the occurrence of a given sequence in the sample. The mass spectrometer looks for the peptide sequences that is selected. 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. In conclusion, the results were positive and the identity of the proteins could be verified 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.