Team:GreatBay United/Engineering
Engineering success
The whole system include three parts: detection system, cascade system and agglutination system.Agglutination system plays a role in showing the detection result. In consideration of the complexity of result presentation form in the existing endotoxin testing methods(recombinant C factor), we decided to use agglutination reaction to visualize the detection result, inspired by blood group test paper and limulus amebocyte lysate.
Design
In order to achieve this，we decided to use yeasts to build our own agglutination system. The first reason is the size of yeast. Compare to other synthetic biological tools, yeast has a bigger size so the agglutination can be more obvious to observe. The second reason is the maturity of the yeast surface display. We can easily use yeast to express the proteins we need to build the system.
Build
We constructed the yeast surface display plasmid based on Vector pYD1.pYD1 is a 5.0 kb expression vector designed for expression, secretion, and display of proteins on the extracellular surface of Saccharomyces cerevisiae cells. Features of this vector allow regulated expression, secretion, and detection of expressed proteins。
The plasmid A (plasmid of Yeast A) is inserted with sequences that code for GFP, HCV substrate and anti GFP(explained below) and the plasmid B(plasmid of Yeast B) is inserted with sequences that code for anti-GFP. Plamid A and plasmid B is transformed into Yeast EBY100 separately. In this way Yeast A and Yeast B are obtained.
Test
In order to verify our assumption in design, we carried out following tests.
1. Verification of the construction of Yeast A&B
We cultured the yeast with YNB-CAA containing 2% glucose at 30℃ and YNB-CAA medium containing 2% galactose at 20℃ to induce protein expression. In order to determine whether the target protein is expressed or not, we add anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) to stain the displayed protein. The fluorescence observation result showed that the proteins are successfully expressed.
2. Verification of the agglutination system design
In our design, instead of expressing GFP alone, Yeast A also expresses HCV substrate and anti-GFP. These three parts are connected together with linkers, as GFP and anti-GFP will bind together on the surface of Yeast A before they meet Yeast B. In this way, Yeast A will not agglutinate with Yeast B as the GFP on Yeast A is inhibited by self-expressed anti-GFP. By adding HCV protease into the agglutination system, the substrate of HCV between GFP and anti-GFP on Yeast A will be cut and the self-expressed anti-GFP on Yeast A will be separate from the GFP and leaving the yeast surface, making GFP be able to bind with anti-GFP on Yeast B and forming agglutination. This is how we use HCV to control the agglutination system, positive and negative results of detection can be present by presenting HCV or absenting HCV in the system.
In the verification test, we mixed same amount of Yeast A and Yeast B together in the buffer, and adding HCV protease into this system.The results showed that the adding of HCV trigger the agglutination between yeasts.
Learn
By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.
Redesign
In our first design, we used anti-GFP-low (anti-GFP with lower affinity) : Part:BBa_K3858013 on Yeast A and anti-GFP-high (anti-GFP with higher affinity) : Part:BBa_K3858014 on Yeast B to ensure our phenomenon of agglutination is obvious. After verifying the agglutination phenomenon can be observed by using this combination, we decided to test the relationship betwwen the agglutination phenomenon and anti-GFP with different affinities.
By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.
Rebuild
Base on Yeast A and Yeast B, we designed 4 yeasts by using anti-GFP with different affinity.
By using the AcLi transformation method, we successfully obtained yeast positive transformers.
By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.
Retest
For the above four combinations of yeast, we did a comparative test on whether to add HCV crude enzyme solution to the system. The results are shown in the following figure. Samples No. 7-10 are samples without addition of HCV crude enzyme solution, showing homogeneous bacterial liquid morphology. Samples No. 11-14 are samples with addition of HCV crude enzyme solution. Compared with the treatment group without enzyme, an obvious coagulation phenomenon appeared in samples No.11-14 after the same time of culture.
The results of the above tests verified that our agglutination system design is reasonable and feasible. These tests had confirmed that the agglutination system can work successfully. When HCV enzymes appear in the reaction system, agglutination will occur, which can be used to construct an artificial Limulus reagent system.