Team:Kyoto/Project/Goal C

Goal C: Characterization of the anti-killer gene

Introduction

We selected SΔTMD1 as anti-killer gene of Lysis box. Lysis cassette encodes S gene, R gene and so on 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.(check Learn more) That is why we selected SΔTMD1 as anti-killer gene.

Then, we checked if SΔTMD1 prevent Lysis cassette from causing the cell death. To research this function of SΔTMD1, we used the E.coli transformed with both these genes( <partinfo>BBa_K358021</partinfo>). Because E.coli is immediately dead if killer-gene is constitutively expressed, we made lysis cassette regulated by lac promoter. The E.coli were grown in the medium without IPTG, and after there were enough E.coli, they were cultured in the one with IPTG to express Lysis cassette. After that, we checked, by measuring the absorbance (A550), if E.coli were alive due to anti-killer gene, SΔTMD1 expressed constitutively.

Method

Bacterial strains

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

• BBa_K358021:Anti-killergene is induced constitutively and killergene is regulated by lactose promoter.
• BBa_K358022:lacP + SΔTMD1RRz, TMD1 is deleted from lysis cassette[SRRz].
• BBa_K358024:This part is the same to BBa_K358021, except for its constitutive promoter[BBa_J23101]. So, this part also induces constitutively SΔTMD1 gene, the anti-killer gene, and lambda lysis cassette[SRRz], the killer gene, is regulated by lacP.

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 culture.

Result

Fig.1.the result of function check of SΔTMD1

Fig.2.Comparison

Fig.3.Lysis cassette ΔTMD1

We measured the cell number of E.coli transformed with <partinfo>BBa_K358021</partinfo> at several time after induction by adding several concentration IPTG. The result is below(Fig.1). This result showed that in the medium with more than 0.03mM IPTG, the number of E. coli is decreasing at a certain point. It demonstrated that In at least more than 0.03mM IPTG, SΔTMD1 can't inhibit lysis cassette completly. Then, we measured the cell number of E.coli transformed with <partinfo>BBa_K358019</partinfo> in the same way, and compared the result of E.coli transformed with <partinfo>BBa_K358021</partinfo> with that of E.coli transformed with (<partinfo>BBa_K358019</partinfo>) to check how SΔTMD1 inhibited lysis cassette(Fig.2). In Fig.2, LacS is E.coli transformed with <partinfo>BBa_K358019</partinfo>, CTLS is the one transformed with <partinfo>BBa_K358021</partinfo>. From this result, we knew in more than 0.5mM IPTG both Lacs and CTLA were decreasing in the number at a certain point, or CTLA was a little delayed decreaing in the number. This demonstrated that SΔTMD1 can't correctly inhibit lysis cassette. So, we doubted whether SΔTMD1 really functioned as anti-killer gene. Although SΔTMD1 has no TMD1 of S gene, SΔTMD1 couldn't show the function as anti-killer gene. Then, in order to check if TMD1 of S gene is essential for this killer gene, we measured the cell number of E.coli transformed with Lysis cassette ΔTMD1, which does not have TMD1 of S gene and that is, encodes also SΔTMD1<partinfo>BBa_K358021</partinfo>, This result is below(Fig.3). From the result, we knew if Lysis cassette ΔTMD1 is induced, the cell death was not caused. It proved that S&Delta ;TMD1 cannot function as killer gene and TMD1 is essential for this killer gene, lysis cassette.

Discussion