Our point-of-care diagnostic gadget must be able to identify both resistant mRNA and bacterial RNA in order to be successful. We require a reporter that produces a signal that is, ideally, both instantly detectable and visible. Therefore, we designed two split ribozymes 1 to detect either the stable endogenous E. coli mRNA hcat 2 and the kanamycin resistance (KanR) of pSB1K3. We combined those with two different reporters: the fluorescent GFP and the chromoprotein eforRED.
We assembled our constructs with Golden Gate Assembly (GGA). To check if our GGAs worked correctly we performed a colony PCR. The reporter and the entire functioning split ribozyme unit make up the amplicon. It is 1700 bp long with GFP and 1661bp long with eforRED. There were a significant number of positive clones in the agarose gels. Figure 1 displays the gel of the clones that were further processed.
The positive clones were further tested using fluorescence and absorbance measurement in a plate reader. After 6 hours of culturing at 37°C, the bacteria were pipetted into 96-well plates. The plates for fluorescence measurement were black with a transparent bottom to minimize crosstalk. For absorbance measurements we used transparent 96 well plates. The bacteria were spun down, the pellet washed in water twice, then the bacteria were resuspended in water and transferred to the plate where a 1:5 serial dilution was performed. The absorbance of eforRED was measured at 230 nm and 582 nm. At 230 nm the platereader only gave values that were over the threshold of detection. At 582 nm, there was no difference between samples with eforRED and without, no signal was detectable. The kanamycin backbone was giving us pink colonies where there should not have been any (figure 2), therefore we were unable to measure the split ribozyme recognizing kanamycin resistance.
The fluorescence of GFP was measured at 485 nm emission and 515 nm excitation. The platereader read a significant amount of the emission laser at 510 nm for the excitation detection, rendering the data useless because all samples—including the negative control—had high values. At 515 nm we could detect just a very small difference between the negative control containing bacteria lacking GFP and the bacteria carrying our split ribozyme GFP construct. The positive control of just the one promoter BBa_J23115 in front of the GFP is very low as well. This shows that the promoter is insufficient to provide a strong signal. The model supported this hypothesis.
In contrast to our split ribozyme constructs with GFP having almost no signal our construct with an inducible GFP provided a strong signal even when uninduced as displayed in figure 4.
Conclusion and Outlook
To produce a point-of-care diagnostic device the split ribozymes need to be transcribed at a higher rate with stronger promoters. Our Model indicated that BBa_J23101 and BBa_J23104 are good candidates. Alternative reporters should be taken into account, as eforRED requires more time to get visible and GFP measurement is limited by a high background fluorescence and the emission laser. For the implementation we also need to consider how to get our system into the bacterium. Phages are helpful for the delivery due to their easy storage and long shelf life. 3 There is still a lot to do, but we are determined to make it happen!
[1] Gambill L, Staubus A, Ameruoso A, Chappell J. A split ribozyme that links detection of a native RNA to orthogonal protein outputs. bioRxiv. January 2022:2022.01.12.476080. doi:10.1101/2022.01.12.476080 [2]Zhou, K., Zhou, L., Lim, Q.'. et al. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Molecular Biol 12, 18 (2011). https://doi.org/10.1186/1471-2199-12-18 [3]Yajie Zhang, Xiujuan Peng, Hairui Zhang, Alan B. Watts, Debadyuti Ghosh, Manufacturing and ambient stability of shelf freeze dried bacteriophage powder formulations, International Journal of Pharmaceutics, Volume 542, Issues 1–2, 2018, Pages 1-7, ISSN 0378-5173.