Cooperator expresses beta-glucosidase with constitutive promoter to degrade cellobiose around it. We had the cell stock of cooperator with pCEL vector containing beta-glucosidase and transformed it into E.coli BW25113. The medium plate contains cellobiose instead of glucose to check whether E.coli that we transformed expresses beta-glucosidase on its surface. The streaking side below shows that cooperator grows on cellobiose containing M9 minimal medium.
[Fig 1.] Cultured cooperator on M9 minimal medium with 0.48% cellobiose
2. GFP expression
Cheater expresses GFP when it obtains glucose. We had the cell stock of cheater with the vector encoding GFP as a reporter gene and transformed it firstly into E.coli DH5α and then to E.coli BW25113.
[Fig 2.] Cultured cheater on M9 minimal medium with 0.48% glucose
[Fig 2-1.] Cultured cheater under fluorescence microscopy showing the GFP activity
As the cheater and cooperator reached OD600 1.8 in M9 minimal medium with glucose concentration 0.48%, we began co-culture in M9 minimal medium with cellobiose concentration 0.48%. Batch co-culture was conducted in 5ml of medium in ratio of cheater and cooperator 1:1, 1:2, 1:4, 2:1, 4:1. 1 was counted as 1ul and each medium was incubated for 1, 3 and 4 days. After each day, we streaked 100ul of the incubated medium on LB agar plate with ampicillin. However, expected bacteria did not grow at all after overnight incubation.
We predicted possible causes of unsuccessful co-culture as follows;
Too little amount of bacteria inoculated in the medium
Low survival rate of pre-cultured bacteria since the optimal OD600 was 1.0 and ours was 1.8 which is thought to be after stationary phase.
Too short period of incubation
We plan to conduct the experiment once again with newly grown bacteria.