Team:Hong Kong HKU/Results
Experimental Results
DNA Nanostructures
Structural Characterisation
The sizes of the 4 ssDNA component strands are shown in lanes 2-5. We confirmed the successful annealing of our DNA tetrahedron on lanes 6-8 in a stepwise manner when adding up ssDNA.
Transmission Electron Microscopy
Our DNA tetrahedral structures are stained and visualised by a Philips CM 100 Transmission Electron Microscope with 100 kV operating voltage.
Since the 3' end of each strand is modified to have a thiol group, gold nanoparticles could then be attached to it. After the strands assemble, all four vertices of the final tetrahedron will be attached by a gold nanoparticle and could be observed under the Transmission Electron Microscope.
Flagellar Over-expression
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).
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.