USTC/SuXiaofeng

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'''(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 [http://2007.igem.org/USTC/BetaGalactosidaseAssay 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.
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'''(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.
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[[Image:USTC_allen11.jpg]] [[Image:USTC_allen11.jpg]]
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[[Image:USTC_ crossrepressiontest.jpg|center|600px]]
*;Key Results
*;Key Results
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*;Difficulties & Overcome
*;Difficulties & Overcome

Revision as of 13:46, 26 October 2007

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
  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.
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.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.

USTC crossrepressiontest.jpg
  • Key Results


  • Difficulties & Overcome