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, 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 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 SδTMD1. Because in these constructs, both the lysis cassette and this allele is regulated by lac promoter and this SΔTMD1 is constitutively expressed, we measured the cell number by the process below using IPTG to research SΔTMD1.

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

Bacterial strains

We used three types of E. coli, E. coli KRX transformed with BBa_K358019, KRX transformed with BBa_K358022.

KyotoFigC001.png
KyotoFigC002.png
KyotoFigC003.png

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.

Result

Discussion