Team:Kyoto/Project/Goal C

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Revision as of 18:40, 25 October 2010

LastModified: 2010-10-25

Goal C: Characterization of the anti-killer gene

Introduction

We checked the function of the anti-killer gene, SΔTMD1. Killer gene, Lysis cassette encodes S gene, R gene and so and the transmembrane domein 1(TMD1) of this S gene is essential for the function of lysis cassette as killer-gene. So SΔTMD1, which is a S mutant with TMD1 deleted, dominant-negatively inhibits Lysis cassette.

Then, we made sure that this TMD1 is essential for lysis cassette and checked if SΔTMD1 functions as anti-killer gene. To check the importance of the TMD1, we checked if a lysis cassette allele with TMD1 of S gene deleted (lysis cassetteδTMD1) functions as killer-gene. To research the function of SδTMD1, we checked if S&deltaTMD1 can prevent lysis cassette from working as killer-gene in E.coli transformed with both these genes. Because, if killer-gene is constitutively expressed, E.coli is immediately dead, we made lysis cassette regulated by lac promoter, after E.coli was grown lysis cassette is expressed by the inducer, and we checked if E.coli were alive due to anti-killer gene, SδTMD1 expressed constitutively .

Experiment 1 Function check of SΔTMD1

Bacterial strains

We used two types of E. coli, E. coli KRX transformed with <partinfo>BBa_K358019</partinfo>, KRX transformed with <partinfo>BBa_K358022</partinfo>.

Measurement

We picked out three colonies from each devices, and cultivated them in M9 medium with 1mM IPTG and without IPTG overnight. Then, we measured A550 of each cultures.

@@ Line 6: / Line 6: @@
 So S&Delta;TMD1, which is a S mutant with TMD1 deleted, dominant-negatively inhibits Lysis cassette.
-Then, after we made sure that this TMD1 is essential for lysis cassette, we checked if S&Delta;TMD1 functions as anti-killer gene.
+Then, we made sure that this TMD1 is essential for lysis cassette and checked if S&Delta;TMD1 functions as anti-killer gene.
-we used E.coli transformed with the lysis cassette allele with TMD1 of S gene deleted to check the importance of the TMD1 of S, and E. coli transformed with both lysis cassette and S&DeltaTMD1 to check the function of S&Delta;TMD1.
+To check the importance of the TMD1, we checked if a lysis cassette allele with TMD1 of S gene deleted (lysis cassette&delta;TMD1) functions as killer-gene.
-Because in these constructs, both the lysis cassette and this allele is regulated by lac promoter and  this S&Delta;TMD1 is constitutively expressed, we measured the cell number by the process below using IPTG to research S&Delta;TMD1.
+To research the function of S&delta;TMD1, we checked if S&deltaTMD1 can prevent lysis cassette from working as killer-gene in E.coli transformed with both these genes.
+Because, if killer-gene is constitutively expressed, E.coli is immediately dead, we made lysis cassette regulated by lac promoter, after E.coli was grown lysis cassette is expressed by the inducer, and we checked if E.coli were alive due to anti-killer gene, S&delta;TMD1 expressed constitutively .
-These E.coli was grown in medium without IPTG and IPTG was added to the culture at proper time.
-The A550 of the culture was measured and the result was compared with that of the experiment of the killer gene in order to find whether the anti-killer gene works correctly.
 ===Experiment 1 Function check of S&Delta;TMD1===