Part:BBa_K2918023
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 292
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 68
Illegal BsaI.rc site found at 94
Overview
This protein is one of the 4 components needed for orthogonal replication
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.
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. The
Mass Spectrometry
In addition to the SDS-PAGE, the identity of the proteins was also confirmed by mass spectrometry. 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
Figure
The peak areas of the resulting mass spectrographs shown in Figure ? reflect the occurrence of a given sequence in the sample. The unique peptides that were screened for were only present in each protein expected to contain the sequence.
Hence, it can be concluded that the results were positive and the identity of the proteins could be verified by mass spectrometry.
In Vitro replication
Toxicity
| None |