In this part, we have deeply thought about the application of our project to the actual situation, and have carried on the thorough exchange with these practical problems and the different specialized direction teacher, summarized as follows:
Bacteria strains:E. coli
Function:Inducibly secretes serine to attract the followers in the system.
Vector Construction:Gene fragments of SerA, serB and serC were amplified by PCR and checked by electrophoresis (Fig. 1A). The predicted sizes of these three cDNAs are 1,233 bp, 969 bp, and 1089 bp, respectively, which matched our experimental results. The vector that expresses SerA, SerB and SerC as an operon is shown in Fig. 1B.
Figure. 1. Electrophoresis of SerA, SerB and SerC cDNA fragments (A) and the operon diagram of pBAD-SerA-SerB-SerC vector (B).M, the DL2000 DNA ladder
Functional Verification:
To demonstrate whether Leader A can secrete serine, we performed serine detection experiment using a serine detecting kit. The results found that the serine levels were significantly increased in Leader A group, as compared to the control group. Moreover, elevated expression levels of serine were detected in Leader A group in a time-dependent manner. These results indicate that Leader A can secrete serine successfully.
Fig. 2 Serine detection result of Leader A
Bacteria strains:Corynebacterium glutamicum(ATCC21885) purchased from the American Type Culture Collection.
Function: It was dervided from wild type and can produce leucine.
Bacteria strains:Starmerella Bombicolapurchased from the China Microbial Culture Collection.
Function: Metabolize fatty acids.
Follower D & Follower E bacteria were tested.
To confirm the system works successfully, we need to verify the theory whether those two kinds of bacteria “Follower D” and “Follower E” have the chemotaxis towards the serine or avoid high concentrations of leucine. Therefore, we made a device consists of needles and capillary tubes (made of medical plastic).
First, to confirm the optimum chemotactic time of serine in this experiment, we put the capillary tubes into the bacterial suspension for 10, 30 and 60 min, respectively. The results found that the samples showed the best chemotactic effect at 30 min (Fig. A). Thus, this collected time point (30 min) is used for all subsequent experiments.
Fig. 5:It was dervided from wild type
Second, we used each needle to absorb different concentrations of 0, 10-5, 10-4, 10-3, 10-2 and 10-1 M serine and the bacterial liquid were smeared in culture plate. As showed in Fig. 2, the number of monoclonal clonoly was increased as the concentration was elevated and reached a maximum at 10-1 M (Fig. B).
Fig. 6:It was dervided from wild type
To further confirm above result, the flow cytometry was used in this project. The number of bacteria was higher in serine group compared with the control group, gradually enhanced from 10-6 to 10-1 M as the concentration was increased and reached a maximum at 10-1 M.
Fig. 7:It was dervided from wild type
Next, we used leucine tubes instead of serine and then calculated the number of bacteria in capillaries for 30 min.
Fig. 7:It was dervided from wild type
To observe the lasting time and final effect of the whole system, we embed Leader A, Leader B and Leader C with sodium alginate and detect the variation of serine, leucine and fatty acid in the co-culture of Leader A, Leader B and Leader C (Fig. 1).
Fig. 9:It was dervided from wild type
The results showed that we can detect the large amount of leucine from Leader B in medium and can also detect the depletion of Leader C to fatty acid.
Fig. 10:Detected concentration of fatty acid in the medium of embedded sodium alginate pellets.
