From 2007.igem.org

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.

• Experimental Design

1. Construction of Expression Library of Lac-Repressor Family [Collaboration]
2. Synthesis of Promoter Sequence with Specific Operators
3. Construction of Low-copy Reporter System with Specific Opertors
4. Selection of Promoter-Repressor Pair(P-R Pair) including re-testing validity of these combination
5. Transfering the Operators to Double Reporter Systems [Collaboration]
6. Quantitative assay of Repression Intensity and Specificity of P-R Pairs
7. 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.

(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

• Difficulties & Overcome