Background

To ensure that our bacteria do no harm to both people and environment, we need to put a set of kill-switch system into the bacteria, so that we can control the growth of them by changing the contents of culture medium.

In our project, we trigger the kill switch of E.coli by adding IPTG into the culture. IPTG can induce the expression of gene coding Limulus anti-LPS factor (LALF) downstream the lactose operator, so that the growth of E.coli will be inhibited.

Principle

Limulus anti-LPS factor (LALF) binds LPS on anti-gram negative bacterial surface tightly, forming a biofilm. This can prevent nutrient from entering the cell, causing the growth inhibition. Also, the biofilm can prevent plasmid spreading to the environment and gene pollution.

Besides, to make LALF cross the cell membrane and bind to LPS, we still need an efficient protein export signal sequence of E. coli to lead LALF into extracelluar environment.

Results

We constructed the plasmid with lactose operator and signal peptide, and transformed this plasmid into E.coli to test if this kill switch do works. We used gradient plate method in our experiment. As you can see in the picture, the concentration of IPTG in the left of the medium is 0.1% while the concentration on the right is zero. We can see colony only on the right of the medium, the kill switch is effective which illustrates that IPTG can induce the expression of signal peptide so as to inhibit the growth of the E.coli. Also, a decrease of OD is observed after we added IPTG to the liquid culture medium.

However ,we haven’t connect the LALF gene with signal peptide yet due to the time limit. We will construct the whole plasmid and transform it into E.coli. to see the sterilization effect of LALF in the future work.