Team:Kyoto/Project/Goal C

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Contents

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 and 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, after we made sure that this TMD1 is essential for lysis cassette, we checked if SΔTMD1 functions as anti-killer gene. we used E.coli transformed with to check the importance of the TMD1 of S and E.coli transformed with to check the function of S&deltaTMD1. 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.


the lysis cassette allele with TMD1 of S gene deleted

lysis cassette regulated lac promoter and S&deltaTMD1 expressed constitutively

Method

Bacterial strains

We used three types of E. coli, E. coli KRX transformed with BBa_K358021, KRX transformed with BBa_K358024, KRX transformed with BBa_K358022.

KyotoFigC001.png
KyotoFigC002.png
KyotoFigC003.png

Measurement

We picked up three colonies from each plate, and cultivated them in M9 medium overnight. The overnight cultures were diluted to 0.1-0.12 (OD600) by M9 medium.After that, we added several amounts of IPTG to the cultures and measured OD600 of each culture at several points.

Result

Discussion