Part:BBa_K2789031
Contents
Plac+RBS+dCBD+OmpA+terminator
This is the standar expression pathway for K2789030. K2789030: Similar to K2789024, this part also displays the surface of the protein. Unlike K2789024, this part of the membrane displays a protein called dCBD (double cellulose binding domain), which allows cells to express this cds to adhere to cellulose.In our project, this part also plays a role in connecting bacteria and algae, because the cell wall component of algae is mainly cellulose, so this part can make the bacteria and algae tightly anchor together to make the structure of the symbiotic biofilm more stable.
Description
We added a lacl promoter and standard RBS as well as double terminator onto the basic part BBa_K2789030 to make it a whole pathway to be expressed.
The instability of biofilm is a big problem in this field currently. This part can improve the stability of symbiotic biofilm of algae and bacteria,
The basic part BBa_K2789030 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 dCBD (double cellulose binding domain), CBD can bind the cellulose and thus it can bind the cell wall of microalgae.
Measurement
We expressed it with OmpA so dCBD can be expressed on the surface of the bacteria. This whole pathway can make our bacteria "stick" to the algae in this way.
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.
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.
We use LB and WT bacteria as control. You can see that this part in both two plasmids have a similar results and a decline of shedding rate in 250r.3min condition. Due to the time limit, we didn’t explore the best condition for the expression and adjust the biofilm formation steps to make it work better. However, although we didn’t do this adjustment, it still worked as expect and did improve the stability of biofilm. A further experiment will be done according to this and we believe it can greatly improve the effect!
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 388
Illegal AgeI site found at 583 - 1000COMPATIBLE WITH RFC[1000]
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