Part:BBa_K2789025

Plac+RBS+lectin-OmpA+terminator

This a a standard expression pathway of K2789024. Lectin is a protein that originally from Green algae. It has positive charge thus can bind with EPS(exopolysubstance),which is secreted by algae. In our project, we wanted our bacteria to connect with the algae to strengthen the symbiotic biofilm. Thus we wanted to make it expressed on the surface of bacteria. We used surface display technology to do that job. The coding sequence of OmpA and lectin had been fused and thus can express the lectin on the surface of the bacteria. Then make it connect with the EPS of algae! You can use this part to bind other agents with negative charges.

Description

we added a lacI promoter and standard RBS and double terminator with the part BBa_K2789024 to express it in a pathway. The basic part BBa_K2789024 can be divided into two parts——OmpA is a normal protein that originally expressed in the membrane of bacteria. Generally we can use this protein fused protein with other protein, which process that can express our wanted protein in the membrane. This process is called ”surface display” and another part of this basic part is lectin, it is originally found in green algae, that can bind with polysaccharide due to its plenty of positive charges. (polysaccharide usually has much negative charges)

We expressed it with OmpA so lectin can be expressed on the surface of the bacteria, and algae usually has a large quantity of polysaccharide in its surroundings, which is called “EPS”, (exopolysaccharide). This coding sequence can make our bacteria “stick” to the algae in this way.

Measurement

To measure how much it can improve the connection between algae and bacteria, we did a series of experiments. Firstly we found the condition of co-culture of bacteria and algae, excluding optimal ratio and culture condition. Then we make it a biofilm by suction filtration method.

Suction filtration device

Biofilm that obtained from suspension system of bacteria and algae

In order to test the stability quantitively, we chose shear force as interference after some failed attempts, the shear force has physical Meaning because it plays that interference also in micro-scale directly to the connection between bacteria and algae, compared with that, shaking or pump don’t. Also, the effect of shear force is uniform to biofilm.

We use this device to create proper shear force by adjusting rotating time and speed.

Blender

We used shedding rate to determine the stability of biofilm and connection strength between two organisms. We put the biofilm onto the inner wall of the cylinder and start stirring the stirrer. After stirring, Collect algae A that remain on the biofilm and exfoliated algae B. Then count A and B separately. shedding rate “B/(A+B)×100% “can be calculated. The number of each organism was counted by very precise methods——chlorophyll method and hemocytometer Counting method. After a series of standard-establishment experiments, we use 250r/s, 3min as a standard because It can better distinguish the level of biofilm bonding strength.

Result

Then we transformed our pathway into bacteria(We actually assembled the pathway into PsB1C3 and PeT28a separately, since the unit is pathway, there should be no dramatic difference between two experimental results) and made it a biofilm with algae. The stability that it improved can be seen by the figure.

The bacteria and algae gathered together in PsB1C3

symbiotic Mass, algae gathered together in group 5 in PeT28a

WT group, no connection between algae (only overlap in microscope)

LB group(no bacteria), No connection at all