Revision as of 17:14, 20 October 2019

AraC-Pc-pBAD-Kid This part is designed mainly for the characterization of the toxin Kid.

In our study, we ought to ensure the toxin encoded within the bistabe system is efficient for killing any bacteria. Therefore, by presenting this composite part with BBa_K3202052 and BBa_K3202054, we provide a tool pack aiming different kinds of bacteria.

Sequence and Features

Toxin

I.Background

Toxin proteins are good materials for detecting leakage of inducible promoters. Because sometimes a molecule of translated toxin, it can have a great impact on cell growth. To further test the leakage effect, we incorporate toxin protein—Kid, ParE and Colicin—into the plasmid. ParE toxins are DNA gyrase (Gyr) inhibitors.[1] Toxin Kid which closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli can inhibit DNA replication. [2] Toxin Colicin binds to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarization of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis.

II.Design

We chose pBAD as the inducible promoter in this experiment for it has lower basal leakage level than XylS. We set all together four different group including one control. We measured OD 600 value which indicate cell growth for the following seven hours.

III.Experiment & Results

At the first level of experiment we incorporate toxins with pBAD&AraC. At the absence of inducer, all of the three toxin experiment group demonstrated a lower OD value with more significant low OD value in BN062（with toxin ParE）and BN065 (with toxin Colicin) than control. Cell growth was affected in each experimental group. When measured after 12 hours, the trend is more obvious with BN065 and BN062 demonstrate a OD value of 0.1891 and 0.1604 respectively. Whereas control group is 0.7755. The result suggests that even without inducer, pBAD system have noticeable leakage when incorporated with toxins which may affected cell growth in some cases.

Figure 1: Growth Curve for Toxin plasmid with pBAD system in the period of 7 hours.

File:T--BHSF ND—Toxin Leakage experiment 2.png

Figure 2: Bar graph of OD 600 value for Toxin with pBAD system after 12 hours.

We want to test our bi-stable system to see wether it can really prevent leakage. We construct three toxin-bistable-system plasmid.When compared to BN063(Kid&pBAD) during a seven hours period, we can observe a significant improvement in cell growth in BN084 as OD 600 value have is higher. This suggests that our system has a significant role in inhibiting the expression of toxin proteins. Later, when we inoculated the colonies, we found that the toxin protein integrated into the bistable state was significantly inhibited in expression. Cell growth was significantly better than BN063.

Figure3: Growth curve for BN063(Kid&pBAD) and BN084(Kid&bi-stable system) in the period of 7 hours.

File:T--BHSF ND—Toxin Leakage experiment 4.png

Figure 4: Bar graph of OD 600 value for BN063 and BN084 after 12 hours.

File:T--BHSF ND—Toxin Leakage experiment 5.jpeg

Figure 5:Colony growth for three types of toxin-bistable-system after 20 hours