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Team:Tsinghua/Experiment1
From 2009.igem.org
Contents |
Synthesis of the Genome of GenSniper
Bottom-Up Approach
In the bottom-up approach, we amplify the biobricks from both the bacteriophage lambda and the adenovirus genome and incorporate them with a given order into molecular cloning vector(s).
Specifically, we choose two molecular cloning vectors to encode two sections of the gene therapy vector genome, pET-28a and pACYCDuet-1.
We incorporate the segment from Nu1 to B on bacteriophage lambda genome into pET-28a while clone the left segment from C to FII (C will be further engineered) into pACYCDuet-1. Before cotranformation with therapeutic DNA, we firstly cotranform the two plasmids encoding the GenSniper genome into BL21 DE3 and evaluate their function.
Top-Down Approach
In the top-down approach, the part of needed segment on the lambda genome will be firstly cutted by restriction enzyme BamHI and HindIII and then incorporated into pET-28a. The lefted segment will be further integrated downstream of the primary clone. Additionally, an expression module for Q protein will be contructed for induction of the expression of the GenSniper genome.
GenSniper Production Evaluation
Firstly, we will spread the cotranformed BL21 DE3 E.coli strains onto Kan-Cm double antibiotics plate to test their compatibility.
Secondly, we will measure the OD changes of the cotranformed E.coli strains to evaluate the function of lysis genes.
Thirdly, we will perform the viroin isolation procedure on our lysed cell culture. Then eletronic microscopy will be applied to observe the package of the GenSniper viroin.
