USTC/SuXiaofeng

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Xiaofeng Su(Allen Su)-Undergraduate of Cellular and Molecular Biology in School of Life Sciences, USTC, P.R. China

XIAOFENG SU

Undergruduate Student of Cellular and Molecular Biology,USTC

Email: allensue@mail.ustc.edu.cn (preference) or xiaofsu@gmail.com

Phone: +86-551-3602469 (Lab)

Mobile: +86-13866722084

Address: Room 439, School of Life Sciences, USTC, Hefei, Anhui, P.R.China, 230026

Research Interest

Directed Evolution for Seeking New Protein-DNA Interactions

Approaches of Synthetic Biology for Forming Novel "Genetic Engineering Machines"

Differentiation and Development of Stem Cells

Research Work

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.

My project description by visual comics: All we need are the specific artificial 'repressor fish' that can definitely bite the specific 'operator hook' exclusively

My Work Basis-By Redesigning lac-repressor and operator. This figure comes from 'Roberto Kopke Salinas, etc. ChemBioChem 2005, 6, 1628 – 1637'

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

(A)The Directed Evolution must be conducted on the base of 2 components-the repressor system and promoter-reporter system.By collaborations with other team members, I've successfully establish these two systems shown as below.

Three kinds of plasmids birthing random mutated repressor called repressor system

Four kinds of plasmids with different specific promoters containing lacZalpha as reporter gene

(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.

The PCR plan for synthesis of specific promoters

The PCR Results

(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.

Figure.7.My arranged selection work desciption

Figure.8.Selection Results-the Red-Mark the colony are a target

Figure.9.retest of my results of P-R pairs

(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.Then quantitative test for repression intensity have been completed by GFP fluorescence microscope assay and Beta-Galactosidase assay for the expression quantity of GFP and beta-galactosidase. But before, we should define a Repression Value that can represent a repression intensity of each binding condition.

File:USTC allen11.jpg File:USTC allen11.jpg

Key Results

Difficulties & Overcome

@@ Line 24: / Line 24: @@
 *;Overall Description
-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.
+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.
 [[Image:USTC_allen1.jpg|thumb|center|500px|My project description by visual comics: All we need are the specific artificial 'repressor fish' that can definitely bite the specific 'operator hook' exclusively]]