Team:Kyoto/Project/Goal C

Goal C: Characterization of the anti-killer gene

Introduction

We checked the function of the anti-killer gene, SΔTMD1. Lysis cassette encodes S gene and R gene and so. The transmembrane domein 1(TMD1) of this S gene is essential for the function of lysis cassette as killer-gene and 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. First, we used E.coli transformed with <partinfo>BBa_K358022</partinfo> in order to check the importance of the TMD1 of S and E.coli transformed with <partinfo>BBa_K358021</partinfo> or <partinfo>BBa_K358024</partinfo> in order to check the function of TMD1. 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.

Method

Bacterial strains

We used three types of E. coli, E. coli KRX transformed with <partinfo>BBa_K358021</partinfo>, KRX transformed with <partinfo>BBa_K358024</partinfo>, KRX transformed with <partinfo>BBa_K358022</partinfo>.

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