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.
Figure. 2. The serine level secreted by Leader A
Bacteria strains:Corynebacterium glutamicum(ATCC21885) purchased from the American Type Culture Collection.(ATCC)
Function: It was derived from wild type and can produce leucine.
Bacterial strains:Starmerella Bombicolapurchased from the China Microbial Culture Collection.
Function: Metabolism of fatty acids.
Follower D & Follower E bacteria were tested.
To confirm the system can work properly, we need to verify the theory that these two types of bacteria “Follower D” and “Follower E” have chemotactic response chemotaxis to serine but show antichemotactic response to leucine. Therefore, we created a device consists of needles and capillary tubes (made of medical plastic) (Fig. 5A). First, to confirm the optimal chemotactic time of serine, we put the capillary tubing with xxxx mM of serine into bacterium suspension for 10, 30 and 60 min, and then determined the bacterium numbers in the tubing. The results indicated that xxx mM showed the highest chemotactic effect at 30 min (Fig. 5B). Thus, we used this time point (30 min) for all subsequent experiments.
Figure. 5. The device for detecting serine levels (A) and the number of bacteria were measured for 10, 30 and 60 min (B).
Second, we used each needles with absorb different concentrations (0, 10-5, 10-4, 10-3, 10-2 and 10-1 M) of the serine to put into bacterial and then smeared them onto LB-agar plates. As showed in Fig. 6A, the colony numbers increased as the concentration rose (Fig. 6B).
Figure. 6 The number of monoclonal colony (A) and the CFU (B) at different serine concentration.
To confirm this result by another approach, we used flow cytometry to determine the amounts of bacteria in this experiment. Consistent with the results obtained above, the numbers of bacteria counted by the flow cytometer also increased in proportional to the concentration of serine.
Figure. 7. The numbers of bacteria measured by flow cytometry (A) and its quantitation (B).
Next, we used tubing filled with leucine, instead of serine, to carry out the experiments and then calculated the numbers of bacteria in capillaries.
Fig. 8:It was dervided from wild type
To determine the retaining time and final effect of the system, we embed Leader A, Leader B and Leader C with sodium alginate and detected the variation of serine, leucine and fatty acid in the co-culture of Leader A, Leader B and Leader C (Fig. 9).
Figure. 9. Different views (A and B) of embedded sodium alginate pellets included Leader A, Leader B and Leader C.
The results indicated a large amount of leucine in medium from Leader B and the depletion of fatty acids by Leader C.
Figure. 10. Fatty acid concentration in the medium of embedded sodium alginate pellets.
