Undergruduate Student of Cellular and Molecular Biology,USTC
Phone: +86-551-3602469 (Lab)
Address: Room 439, School of Life Sciences, USTC, Hefei, Anhui, P.R.China, 230026
- Directed Evolution for Seeking New Protein-DNA Interactions
- Approaches of Synthetic Biology for Forming Novel "Genetic Engineering Machines"
- Differentiation and Development of Stem Cells
- Overall Description
Unlike the real wires of electrocircuit board, in cytoplasm, chemical signaling molecules in an relatively open systems. For obtaining the signaling transduction parts of repression with high fidelity, I've experimentally designed and acquired some specific repressor-promoter pairs(R-P pairs or P-R pairs)based on Lactose Operon by directed evolution on plate. Besides, through quantitative assay,the novel artificial R-P pairs I selected have been tested for their binding performance so that P-R pairs of highest affinity and specificity can make a figure out of P-R pair candidates. Most of the parts in my work have been BioBricks-Standardized and work as BioBrick parts.
- Experimental Design
- Construction of Expression Library of Lac-Repressor Family [Collaboration]
- Synthesis of Promoter Sequence with Specific Operators
- Construction of Low-copy Reporter System with Specific Opertors
- Selection of Promoter-Repressor Pair(P-R Pair) including re-testing validity of these combination
- Transfering the Operators to Double Reporter Systems [Collaboration]
- Quantitative assay of Repression Intensity and Specificity of P-R Pairs
- Results From RM(Repression Matrix) to ORM(Orthogonal RM)
- Work Process and Summary
(B)Within the work of establishment of two selection systems, a time consuming step is synthesis of target specific promoters that contain the mutated site design by Do. On account of economic and convenient aspect, I use 2-Step unparallel PCR by 3 fragments of primers and 2 times of PCR with different reaction conditions.As Below.
(C)I've develop the selection work by the means of Blue White Selection on top agar Luria-Bertani broth to obtain the R-P pairs.Below are some pictures from my experiment design and experiment work.
(D)I've conducted a 'cross repression' test by transforming the selected repressors to their specific promoter-reporter system and other non-specific promoter-reporter systems.We selected 7 repressor-promoter pair candidates from Blue/White Screening results above for quantitive assay of specificity and affinity. In addition, 2 existed represor-promoter pairs are added to this work as new candidates. Then, each repressor-expression plasmid is transform to each Top10 competent cell with specific target promoters. Eventually, each expression quantity of LacZ alpha or GFP is measured by ONPG assay(LacZ) or fluorescent assay(GFP).The process are shown as below.
- Key Results
The consequent data of reporter's expression is, by formula, converted to the Repression Value(R.V.) representing the repression intensity of each repressor-promoter pair. From the formula below we can see higher value of R.V. represents the higher expression of reporter gene and indicates a lower repression while the reverse represents a high repression. The sets of R.V. is depicted on the scheme below which have been transformed to corresponding Repression Matrix-more visual that the coordinate scheme. And the Orthogonal Repression Matrix birthing from Repression Matrix can be used to acquire specific repressor-promoter pairs with high specificity.
a) Repression Matrix
Performances of our wires are shown as so-called "Repression Matrix", an array of R.V. with variant combinations of artificial repressors and operators. A "Repression Matrix" taken from the literature and uniformed is also plotted in this page.
b)Orthologal Repression Matrix
Several 'wires' without interference are selected based on that repressors can only specifically bind to the right promoter without strong repressed interaction with other promoters which have their own special repressors. Thereby, the red color plot in Repression Matrix should be placed on the right site by interchange of columns so that each row and each column in the Matrix can have only one red plot. The other repressor candidate columns which cannot pass muster will be deleted from the RM. Eventually, a 3x3 Orthogonal Repression Matrix for the demonstration system shown in Figure 9, which looks like an orthogonal matrix in mathematics, contains single red plot in each column & each row, reflecting the specific repression.
- Repressor Design in silico
Besides my artificial design work in experiment, we also conduct a program for screening the target repressor-promoter pairs via computational biology mainly by Ding Bo, shown as the following website: Repressor Design in silico
- Difficulties & Overcome