Team:HK SKHLPSS/Characterization

Characterization


Characterization

p1

In this characterization part, we would like to check for the activity from the following parts: BBa_K2054001 and BBa_K2054005 designed by 2016 HKU iGEM Team. These two parts are responsible for the formation of Guanine-riched quadruplex. The single-stranded DNAs produced were used to do the same ABTS peroxidase assay to check for the presence of the G-quadruplex with reference to their protocol.

Other than hemin, ABTS and hydrogen peroxide, these tubes contain different mixture. Details are shown below:

  • Tube A: No parts and target sequence
  • Tube B: With nano-structure comprised from BBa_K2054001 and BBa_K2054005 but no target sequence
  • Tube G: With nano-structure comprised from BBa_K2054001 and BBa_K2054005 and target sequence
  • Tube H: With nano-structure comprised from BBa_K2054001 and BBa_K2054005 and random sequence.

Primary data:

p1

Result: Check for the peroxidase activity of the nano-structure formed from the parts

p1

From the above graph, it is found that when there is only the parts but with no target sequence (Tube A), the absorbance was at 0.104. On the other hand, the presence of the target could give a high absorbance at 0.176, indicating that their nano-structure comprised by these two parts could effectively detect the presence of the target sequence. On the other hand, when nano-structure is mixed with random sequence, low absorbance resulted, indicating that the nano structure formed from these two parts has a high specificity of the target sequence.

The next experiment involved using the nano-structure comprised from BBa_K2054001 and BBa_K2054005 to detect the target in different concentrations, and to check whether there is a relationship between the concentration of the target sequence and the absorbance.

Result: Check for the peroxidase activity of the nano-structure formed from the parts against different concentration of the target sequence

p1

From the graph, it could be found that the absorbance increased with increasing concentration of targets and they were in a linear relationship, showing that the nano-structure formed from these two parts are applicable to detect the concentration of target sequence. The limit of detection is 7.4%, showing that the absorbance is highly specific to the concentration of the target sequence.

All in all, the nano-structure worked as long as the concentration of target sequence is higher than 37nM. So this designed nano-structure from these two parts could work reliably and is highly specific as the absorbance in tube H (with random sequence) showed a low absorbance. Hence, the two parts worked as per designed.

p1