Detectives' Journal

Sept 1 - 12 Reporter/Repressor Genes Restarted efforts to ligate a reporter or repressor to our synthesized construct. Transformed E. coli with the synthesized parts, this time done properly with IPTG and X-gal on the agar plates - only few blue colonies were shown indicating that transformation was highly successful. Colonies were selected, used to make overnight cultures, from which plasmid DNA was isolated via the plasmid miniprep protocol. The C2-P22 repressor was ligated with the Pveg_C1-lambda_consensusRBS construct, using both single-operator and triple-operator variants. Likewise the RFP reporter was ligated with the Pveg_C2-P22_consensusRBS construct with both single- and triple-operator variants. The RFP-ligation allows for straightforward visual screening as successful ligations are indicated by red colonies. The repressor-ligations are being screened via colony-PCR.

Isothermal PCR We used the isothermal PCR kit for the first time and discovered a new problem - the PCR product contains protein-bound DNA which prevents visualization on agarose gels, and may pose a problem for transformation if DNA is not free. DNA pruification is required, however, fortunately dissociation of protein-bound DNA may be induced via 1/10 dilution of the PCR product. This is promising as we would expect a similar dilution in our final prototype, we expect that the strong association between DNA and protein will not pose a problem.

The TwistDx RPA isothermal PCR kit was originally designed for rapidly amplifying short (<400bp) amplicons, unfortunately for our purposes we require amplification products to be closer to 1kbp in size. According to literature ( Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA Detection Using Recombination Proteins. PLoS Biol 4(7): e204.) it is possible to amplify sequences of up to 1.5kbp using RPA. We are attempting to optimize conditions so that we obtain longer amplicons. We may adjust parameters such as the concentration of primers, magnesium-acetate, dNTP, and ATP, as well as incubation temperature - with the aim of increasing the elongation time while providing enough resources (i.e. ATP) to allow for a longer reaction time.

Blood PCR & Hybrid PCR (isothermal + blood) Blood PCR has shown to work wonderfully, with PCR products formed at a blood concentration of 20% v/v; the KAPA blood PCR kit used claimed to have optimal efficiency up to 10% blood v/v. The blood-compatibility is conferred by a second-generation Taq polymerase, engineered via directed-evolution, that is resistant to polymerase inhibitors typically found in blood. For comparison, PCR with typical first-generation Taq showed drastically reduced efficiency of amplification at blood concentration of 1% v/v.

The next step was to show that isothermal PCR (RPA), supplemented with the blood-compatible Taq polymerase, would work efficiently even in the presence of blood. 25, 50, or 75% of the volume of the isothermal PCR reaction (i.e. the buffer and water) was substituted with the KAPA blood PCR mix. Results showed that indeed the blood PCR kit supplement allowed isothermal PCR to work in the presence of up to 10% v/v of blood, when 25% of the volume was by the blood PCR mix. Unfortunately at 50% and 75% substitution, the reaction did not work at all, this is most likely due to a lack of ATP - the isothermal PCR buffer contains ATP necessary for recombinase activity, at higher substitutions of blood PCR mix the buffer was not used or was not enough in regards to the amount of ATP present.

We are also working on determining the sensitivity of our amplification. Results will be judged by visualization on agarose gel, if no other means of detection of DNA amplicons are available. Sensitivity will be quantified using the standard unit of CFU/mL.