Team:USTC-China/Methods

General IdeasOur Defences Designed

Some description of the picture

As we conceived,our new bacteria need to have appropriate defences in order to prevent the whole population from phages.Generally, defence can be classified as active defence and passive defence. If we make our engineered bacteria defend actively, they will expend large amounts of energy fighting against the phages even in an environment without phages. If so, the production of fermented products will be affected. For this reason, we successfully design our bacteria which can detect the infection of the phages. Only when the phage infects the host, the host will start its resistance against the phage.

We kill the phage by means of making the host suicide. Provided that the host can suicide fast enough and die before newly assembled phages become mature, the new waves of infection will be successfully avoided. In this case, the damage the phages can cause will be much lower. What’s more, our engineered bacteria can efficiently kill the lysogenic bacteria which hide in the colonies. This will guarantee the colony’s safety from the origin.

However, the phage is still possible to release plenty of mature phages before the host suicides. What’s worse, the circumstance in the fermentation tanks is very beneficial for bacteria to grow. And when a lysogenic bacterium lives in a fertile circumstance, the phage tend to transform into lytic life cycle.(See the introduction about lambda phage and its lysogenic and lytic life cycle). In order to prevent the phage from reproducing and releasing large amounts of newly assembled phages, we must decrease the possibility for the phage to turn into the lytic life cycle and make it stay at lysogenic life cycle. In this case, we can win plenty of time for this bacterium to defeat the phage.

At the same time, to keep the safety of the colony better, we use the quorum sensing system to make the bacteria around the host start to defend. But due to the complexity of the E.coli’s membrane, we failed to find methods to prevent the phages from invading into the bacteria. Also because of deficiency of time, we did not complete the quorum sensing system.

Our Methods In DetailAs I thought before

Here is the gene circuit we designed

We use the promoter pRM(originally from the genome of lambda phage) to detect whether there are phages invading the bacteria. If a phage infects a bacterium, the protein CI which the phage produces will bind the site OR1 and OR2. Thus, the pRM will be activated and the products downstream the pRM will be expressed. What’s more, the protein Cro cannot inactivate the pRM since our pRM has been modified. There are only sites OR1 and OR2 but no OR3. All the genes we use for resistance to phages are downstream.

We use the lysis as a suicide gene to kill the lambda phage and its host together. The lysis protein causes modifications of inner membrane. Then it activates OmpLA to destroy the outer membrane, which will result in cell death. When the lysis protein reaches a critical concentration, the death effect occur. If the phage cannot turn into lytic life cycle or newly assembled phages are still immature, no waves of infection will occur afterwards.

We use the antisense RNA to prevent the phage from turning into lytic life cycle. Downstream of the pRM there is a cro gene which is ligated reversely without a RBS ahead of it. This gene can express antisense RNA (we call it anticro) which can complementarily bind with the mRNA of cro. If the mRNA is bound by the anticro, it cannot translate the protein Cro and will degrade soon. In this case, the amount of Cro will decrease and repressor CI will make lambda phage stay at the lysogenic life cycle.