Part:BBa_K1698001:Experience

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Applications of BBa_K1698001

Part shown with Primers for the creation of 30bp HPV Detector and 55bp HPV Detector. Enzymes used to digest the detector in construction are also indicated.

UCC iGEM 2014 developed the first method of detector construction by digestion using 4 enzymes (Kpn I, HindIII & nicking enzymes, NtBsI & NtBsQI) (click here to read our protocols). Results of this method of construction were generally positive as positive control plates achieved colonies TNTC and negative control plates showed <10 CFUs. As seen in figure 1 and 2, showing detector reactions using digested detector made by UCC iGEM 2014, tested in June 2015 & September 2014, negative plates showed CFUs. This may have been due to the presence of undigested detector that transformed without target needing to be present. To improve on this, Cork iGEM 2015, created a new protocol for detector construction by PCR.

Results generated by UCC iGEM 2014 relating to HPV Detector constructed by digestion. Negative plates show a number of CFUs. The new protocol (click here to read our protocols) involves using primers specific to the detector part and amplifying this part on a plasmid containing GFP biobrick and chloramphenicol resistance. These primers are illustrated in Figure 2. Further modification of the detector is then carried out to activate. A DpnI digest after PCR ensures that template DNA is digested, being rich in GATC restriction sites. A subsequent clean up step ensures that template DNA is removed to minimise re-annealing to detector. Next, an enzyme digest was carried out using the same 4 enzymes as the original protocol (NtBsQI, NtBsI, KpnI & HindIII), shown in Figure 2. This was done to create single stranded overhangs complementary to the target. The detector was then reacted with decoy oligos (complementary to the top strand of the detector, which is to be removed) in excess to yield the single strands of the detector. Another cleanup was then carried out to remove the decoys. Further experimentation showed that the decoy oligo step was unnecessary and the detector was ready for use after the 4 enzyme digest and PCR cleanup.

Figure Legend - Results of detector reactions using the PCR constructed detector showed less background overall. However, the number of CFUs achieved on positive control plates also showed a reduction. This may have been due to DNA concentration in the final detector sample being less after multiple clean ups during construction. This may be seen in Table 1 which shows a comparison of detector reactions carried out simultaneously using digested and PCR constructed detectors.

Figure Legend - Results of transformation using highly competent cells and detectors (PCR Constructed (left) & Digested detector (right) in the presence (top) or absence (bottom) of the target sequence.

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