Latest revision as of 09:33, 21 October 2019

flhDC transcription factors (Salmonella)

The gene is flhD and flhC transcription factors. Upon expression of the transcription factors, it would up-regulate the expression of flagella in Salmonella Typhimurium.

Sequence and Features

Introduction

Biology

Part structure

The part for Flagella Production Boost

BBa_K3063000 & BBa_K3063111 The BioBricks encodes flhD and flhC (flhDC) transcription factors for over-expression of flagella in Salmonella Typhimurium. Upon expression of the transcription factors, it would up-regulate the expression of flagella in Salmonella Typhimurium and facilitates locomotion. A lac promoter is incorporated into the BioBrick for driving expression of flhDC in S. Typhimurium.

Part Therapeutics

Salmonella as an oral therapeutic bacteria has gained a lot of attention and intensive research thorough the years. ^[1] Its ability to accumulate at intracellular region and colonise at tumour region make it a perfect candidate for the vector of DNA vaccines. Research showed that the transcription factor flhD and flhC behind the production of flagella is the key reason of Salmonella special ability. ^[2] Hence, we postulated that with the over-expression of flhDC transcription factor, the tumour-colonising ability could be boosted and the efficiency of therapeutic transfer could be enhanced.

Characterization

Colony Forming Units (CFU)

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, the content of the spheroid were diluted and spread on a plate for Colony Forming Units (CFU) measurement.

The results show that the transformed S. Typhimurium which expressed a lot more flagella form much more colonies than the normal S. Typhimurium. Because increased number of flagella will increase the motility of Salmonella and thus they can move faster and form more colonies.

Cloning

To drive the over-expression of flagella in Salmonella Typhimurium, we constructed two BioBricks: 1) flagella transcription factor (flhDC) and terminator; 2) flagella transcription factor with both the lac promoter and terminator.

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).

References

1. ↑ Darji, Ayub, et al. "Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier." FEMS Immunology & Medical Microbiology 27.4 (2000): 341-349.
2. ↑ Raman V, Van Dessel N, O'Connor OM, Forbes NS. The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. J Immunother Cancer. 2019 Feb 12;7(1):44. doi: 10.1186/s40425-018-0490-z.