In harnessing genetic and biological engineering for our overall project goals, we undergo smaller iterations of the engineering design process. From building and testing circuits, to assays, to engineered organisms, we learn from, and consequently iterate, each design until the optimal one is reached. Below we describe two different cycles we went through in our wet-lab, where we learned from our own designs and tweaked them accordingly to reach more desired results.
The iterations of our genetic circuit’s design differed in the way they implemented the AND-gate system to ensure more sensitive readout of immune activity.
While the biomarkers and hence the inducible promoters were the same as the current workflow, with our initial design, the activation of the sicA promoter resulted in production of a lactate permease to transport lactate into the cell, which would then activate the lactate-inducible promoter. In turn, the activation of this promoter would express luciferase with a secretion tag, producing our reporter for secretion in urine.
1) Produce a stable line of bacteria that have their endogenous lactate transporters knocked out by CRISPR
2) TNFa induces the activation of the sicA promoter which allows for the prosecution of lactate transporters
3) If lactate is present in the extracellular environment, it enters the cell through the transporters and activates the lactate-inducible promoter, leading to the production of nanoluciferase with a Salmonella secretion tag
4) The nanoluciferase enters the bloodstream and is detected in the urine
After designing this circuit and seeking feedback from our graduate advisors, we uncovered various fundamental issues with the setup and logic:
- Salmonella naturally takes up lactate as part of its metabolism(Gillis et. al, 2019), which acts as an integral nutrient source. Hence, each cell must already have a way of transporting lactate through its cell wall, likely through theexistence of lactate permease in its membrane. Knocking out lactate permease, as was proposed in our original design, would therefore interrupt Salmonella metabolism and possibly interrupt normal function of our chassis, or even be lethal to the cell.
- Luciferase secretion by the secretion tag we designed would release the protein into a host cell rather than the extracellular matrix.
The solution to this concern would be to avoid knocking out lactate permease. However, endogenous transport of lactate into the cell abolishes the AND-gate concept in our system since only lactate would be required to produce the reporter. Since lactate is already naturally being uptaken by the cell, this results in the constant production of our reporter in lactate-rich environments, even without the presence of our second biomarker. As explained in our Design page, given that lactate on its own is not a strong enough indication of tumour presence, our system would not be a very reliable tool for characterizing immune activity in tumours without the AND-gate.
Since these issues are all caused by making lactate permease mediate the AND-gate, we decided to produce a system that would not require this transport protein but keep the AND-gate. It would instead follow the logic below:
1) Produce a wild-type Salmonella cell line that expresses both constructs with TNFa-inducible promoter and the lactate-inducible promoter.
2) The construct with the TNFa-inducible promoter would drive expression of components luxA, luxB, and luxC of the lux operon, and the lactate-inducible promoter would drive expression of components luxC, luxD, and luxE of the lux operon.
3) Since luciferase is produced by luxA and luxB, only when both TNFa and lactate are present will all components of luciferin be produced. Split-protein systems rely on hydrophobic forces automatically bringing both halves back together. Since hydrophobic residues are usually protected within the interior of the protein, but become exposed when split, it becomes energetically favorable to rejoin the split halves of the protein to prevent hydrophobic interactions with polar molecules. Hence, once all components of luciferin are produced, the full protein will assemble and luminesce, producing the reporter only in the presence of both biomarkers.
In conclusion, the genetic circuits we constructed, which were the central base for wet-lab efforts, went through a redesign that stemmed from careful consideration by undergraduate and graduate advisors.
LACTATE INDUCTION EXPERIMENTS
As the lactate-inducible promoter gets induced once lactate binds to its substrate, we measured the promoter's activity by testing different concentrations of lactate with a plate reader. On each run of our induction experiment, we learned from the previous trial run and built upon its design to reach the ideal experimental setup.
We designed the experiment to be performed on a Varioskan plate reader and a 96 well plate.
Multiple different strains of cells were used to ensure the integrity of our experiment. Our construct of interest (pLactate + lldR + RFP plasmid) was transformed into chemically competent E. coli and screened with chloramphenicol. The construct was built as described in the circuit construction section of our Experimental design page. As a positive control, we used a constitutively expressing RFP cell strain to determine the ceiling of possible fluorescence from our lactate regulated construct. For a baseline/negative control, we used an E. coli cell strain with an “empty” vector (contains a pSB1C3 plasmid without an insert). Another negative control for the plate was a lane of “only media”. The cells were to be seeded into the wells at a cell density of 0.1 OD600. The cell concentration would allow us the ability to see the growth of the cells while shortening the time it took for the cells to reach the stationary phase
We chose specific lactate concentrations for the media we were using. These concentrations were to elucidate the viability of our construct in identifying healthy tissue vs tumorous tissues. The values were determined through lactate measurements in literature. We settled on 0mM, 2mM, 5mM, 15mM, 40mM and 100mM lactate since these values gave us good enough separation to determine the viability of our construct. Since the plate and plate reader worked best at a volume/well of 200-300 uL, we decided on a total volume of 250 uL (200 uL media, 50 uL cells). The media would be plated first, and then the cells added. Concentrations would be calculated to take into account the lactate dilution factor that would happen after the cells were added to the media (e.g instead of plating LB media with 100 mM lactate, 125 mM would be made instead). Similarly, this dilution factor would be taken into consideration for cell concentrations. Both of this would allow us to keep the concentration of lactate and cells to be properly controlled.
On the day before the experiment was run, we inoculated RFP cells, cells with an empty plasmid (e.g. plasmid pSCB1C3 without an insert), and the cells with the construct (lactate inducible promoter system and reporter protein gene). Then, we made and diluted the lactate solution to the appropriate concentrations to be put in the reader.
On the day of the experiment, we first prepped the inoculations. Using the spectrometer, we diluted the cells to an OD600 of the cells to 0.5 OD600. Then, we loaded our media and our cells into a 96 plate using a multichannel pipettor, and ran the reading of the plate. The plate reader was run for 18 hours overnight and the data was collected the following morning.
Upon discussing results with our advisors, we added a few improvements for our second lactate induction experiment run. First, we made sure to wash the cells in LB before employing them in our experiment. Washing the cells enables us to remove certain metabolites that may have accumulated overnight and prevent them from interfering with our cells. This was done through pelleting down the cells and resuspending it with fresh LB (repeated twice). Second, we ensured to add an OD read in our plate reader cycles so that the fluorescence reads could be normalized to the cell growth/population. Third, we switched out our clear, white plates for clear, black plates for more accurate fluorescence readings.
In this iteration, the OD600 reads allowed us to normalize the fluorescence reads. After analysis, this returned a more expected pattern where fluorescence increased with lactate concentrations, proving that our construct does work. Early on however, bubbles at the start (0 h timepoint) skewed our initial reads, which is something we need to take into account next time.
A large part of our proof-of-concept wet-lab work would be to corroborate that our construct works in the chassis (S. typhimurium) of our choosing. With this in mind, we transformed our construct, along with a RFP-containing plasmid and an "empty" vector, into electrocompetent Salmonella cells. We then ran a similar induction experiment to those outlined above. Learning even more from our second experiment run, we now understood that bubbles produced from pipetting introduce early experimental artifacts. We also took on additional lactate concentrations (0. 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 100 mM lactate) and decreased the times between our runs (1 hour to 10 mins) to increase the overall resolution of our data from the experiment.
A large part of our proof of concept wet lab work would be to corroborate that our construct works in the chassis (S. typhimurium) of our choosing. With this in mind, we transformed our construct, along with a RFP containing plasmid and an “empty” vector, into electrocompetent Salmonella cells. We then ran a similar induction experiment from the ones outlined above. Learning even more from our second experiment run, we now understood that bubbles produced from pipetting introduces early experimental artifacts. Taking this into account, we made sure to start incubating and reading after the bubbles disappeared. We also took on additional lactate concentrations (0. 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 100 mM lactate) and decreased the times between our runs (1 hour to 10 mins) to increase the overall resolution of our data from the experiment.