Team:Hong Kong HKU/Co-culture Optimization
Co-Culture Optimization
Flagellar Transcription Factor Expression
The co-culture system was built and modified according to a co-culture system developed at 2019 [1], utilising 2.5 µg/ml of gentamicin in the non-antibiotic supplemented cell culture medium 2 hours after clearance of extracellular after bacterial incubation. However, lower bacterial number with shorter incubation time could be utilised in our improved co-culture system, with our flagellar transcription factor biobrick.
To drive the over-expression of flagella in Salmonella Typhimurium, we constructed two BioBricks: 1) flagella transcription factor (flhDC) gene amplified from the Salmonella genome; 2) flagella transcription factor with both the lac promoter and terminator. The lac promoter acts as a constitutive strong promoter in salmonella due to the lack of lac repressor, also shown in our part characterisation.
To test whether we successfully cloned the constructs into the vector, colony PCR was done on 2 rounds of sub-cloning. First, the sequence encoding flhDC was inserted into pSB1C3. Secondly, the fragment containing both the flhDC and terminator was cut and inserted into the final vector containing lac promoter. The results of colony PCR show the successful cloning of two BioBricks.
Lanes 2-4 are triplicates of the correct constructs containing the lac promoter, flhDC and a terminator with matching sizes (1582bp). Lane 5 contains the PCR product of the intermediate constructs with only the flhDC sequence and a terminator with matching sizes (1382bp).
To test whether the construct works and its effect, the BioBrick containing the lac promoter, flagella transcription factor flhDC sequence and terminator was transformed to Salmonella Typhimurium.
The transformed S. Typhimurium were then cultured inside a liver cancer cell spheroid of Huh7 cell line. After incubation for 24 hours, the spheroids were washed with PBS for several times without disrupting the physical integrity. And the content of the spheroid were broken down by repeated pipetting, diluted and spread on plates for Colony Forming Units (CFU) measurement.
The results show that the transformed S. Typhimurium which expressed a lot more flagella form approximately 10-fold more colonies than the normal S. Typhimurium, which also was in consensus with the original research utilising modelling for tumour environmentsRaman, V., et al., The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. Journal for immunotherapy of cancer, 2019. 7(1): p. 44.. It successfully proves the increase in accumulation of salmonella in spheroid co-culture systems by utilising a single simple biobrick, leading to development of more mature spheroid co-culture systems .
This is the video showing normal Salmonella Typhimurium without flhDC transcription factor. The salmonella colonise and accumulate around the cancer cells and show normal activity.
This is the video showing Salmonella Typhimurium transformed with the BioBricks encoding flhDC transcription factors. The flagella of S. Typhimurium are thus over-expressed and increased motility and activity. As shown above in the video, the salmonella around the cancer cells have higher activity than that of normal Salmonella as they seem to swim faster at the edge of cancer cells.
This video shows the activity of Salmonella around liver cancer cells. The orange signal indicating the presence of RFP coding sequence come with the BioBricks. The results show that
1. Both bacteria species could colonise around cancer cells but only Salmonella can perform bactofection and enter cancer cells and accumulate.
2. Co-culture system where both liver cancer cells and salmonella incubated together is achieved by Huh7 spheroid incubating with engineered Salmonella. This co-culture system can reached 100% spheroid colonisation. As the co-culture system adopted in our project utilises 2.5 ug/ml of gentamicin [1]
3. Based on the videos and photos, the loss of RFP-tagged plasmid outside tumour diminish and reduce in number much more quickly than that inside the tumour cells. It proves that the entry of Salmonella into tumour makes the plasmid stay longer.
Reference
- Harimoto, T., et al., Rapid screening of engineered microbial therapies in a 3D multicellular model. Proceedings of the National Academy of Sciences, 2019. 116(18): p. 9002-9007.