Characterization:

Cell-Free Experiments

Prior to any experimentation, we created 100 µL standard curves using the iGEM Measurement protocols to allow us to convert any data converted our data from arbitrary absorbance and fluorescence units to MEFL/particle.

After we created our standard curves, we decided to measure one of our past team’s composite parts from 2017, PLas_sfGFP Part BBa_K2522001which produces sfGFP in the presence of N-3-oxododecanoyl homoserine lactone (3OC12) to fulfill our Bronze Medal Criterion. Please see our Measurements page for further detail.

Since we were unfamiliar with working with a cell-free system, we wanted to work with optimizing a circuit that we were familiar with: PRhl_GFPa1.The plasmid was used by our team last year and senses and responds to the quorum-sensing molecule N-butanoyl-L-Homoserine Lactone (C4-HSL). The J23117 native promoter constitutively expresses RhlR, which activates the PRhl promoter site when bound to C4-HSL allowing for the production of the Green-Fluorescent Protein (GFPa1).

Prior to testing it in a cell-free system, we wanted to confirmed its functionality in cells and set up overnights and graphed the results below.

Having confirmed functionality, we moved on to testing it in cell-free.

Since our chromium circuit would contain T7 promoters and native promoters, we tested PRhl-GFPa1 in a T7-optimized kit and looked at the effect of adding bacterial polymerase. Unfortunately, we saw no signal in our PRhl-GFPa1 circuit but did see a signal in our positive control pY71-sfGFP.

We tried to optimize PRhl-GFPa1 in cell-free again but this time we used a native promoter-optimized kit and adding T7 RNA polymerase for our control. We did not see any signal expressed in both the PRhl-GFPa1 plasmid and pY71-sfGFP.

Troubleshooting Cell-Free

We decided to begin troubleshooting our cell-free system and began by testing the functionality of the positive control (pAME215) which has a native promoter and will constitutively express a green fluorescent protein in both the T7-optimized kit and native promoter-optimized kit.

As shown in the graph above, the T7-optimized kit yielded higher results and will work with both types of promoters.

We proceeded to investigate different concentrations of DNA in cell-free and concluded that pAME215 functioned in a T7-optimized kit, however, PRhl-GFPa1 (labeled C4-HSL) did not produce any signal regardless of concentration.

We also wanted to determine whether DMSO, which is what C4-HSL is dissolved in, was potentially affecting our cell-free reaction. Based on the results graphed below, DMSO had no effect on our reactions.

pY71-sfGFP

Due to time constraints and since our PRhl-GFPa1 was not functioning in cell-free, we moved on to drying down cell-free reactions of our pY71-sfGFP positive control which we had previously confirmed function in cell-free. We also wanted to determine if adding DNA into the reaction or rehydrating with DNA improved functionality, and if increasing the volume of the reaction increased yield.

We concluded that adding DNA before in a 25 uL reaction yielded the highest fluorescence.Since we had a success lyophilization, we moved on to applying our circuit on a paper sensor and after one hour of incubation, we saw fluorescence.

pY71-bFMO

After receiving the pY71-bFMO plasmid from Lab 2, we confirmed the functionality of it in cells and concluded that with the addition of tryptophan, there was a greater yield of indigo.

We proceeded to test in cell-free but there was no signal detected.

Since pY71-bFMO was not functional, we took a look at our sequence and discovered that there was an illegal XbaI cut-site in between our T7 promoter and RBS region.

===J18912-sfGFP=== ===J18912-bFMO===

The circuit responded to 3OC12 in a dose-dependent manner. We began seeing signal at 10-4 μM and the signal saturated at 10-1 μM. The sensor showed a 60-fold increase in signal upon activation based on the non-linear fit analysis. Analysis was done on GraphPad Prism 5.

We have submitted this characterization in order to fulfill our Bronze Medal Criterion, although we have also used these measurement protocols for the remainder of our experiments. However, our standard curves differ based on factors such as volume and plate type. We’ve graphed all of the other standard curves below.

J18912-sfGFP

We created six overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and either E.coli DH5α cells containing J18912-sfGFP (three overnights) or either E.coli DH5α cells containing pY71-sfGFP(three overnights) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.

Through these overnights, we were able to confirm the functionality of both plasmids, and we measured the fluorescence and absorbance of each overnight by performing an endpoint read of each overnight in a 96-well flat bottomed plate. In order to obtain data in the range of our standard curve, we diluted the overnights two-fold by mixing 50 µL of each overnight with 50 µL of LB.

We then used the 100 µL standard curve we created using the iGEM Measurement protocols and converted our data from arbitrary absorbance and fluorescence units to MEFL/particle which we graphed below.

Cell-Free Experiments

After confirming functionality in cells, we moved on to optimizing our system in cell-free. We set up cell-free reactions and also Although, in cells, there seemed to be no difference in signal of our two plasmids, in cell-free, there was a difference, as shown in the graph below. We believe that this most likely resulted from the difference in DNA concentrations which affects the cell-free system.

We also lyophilized our cell-free reactions, and rehydrated them with 25 µL of water. After reading them on the plate reader for four hours, and graphing the data, we were able to confirm functionality of both plasmids, although there was a lower signal in the lyophilization.

Paper-Based Sensor

Since both of our plasmids functioned in cell-free, we moved on to applying our plasmids on our paper-based sensor. After one hour of incubation at 30 °C, we were able to confirm functionality and see signal for both plasmids.

J18912-bFMO

We created three overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO and three overnights that contained 2.4 mL of LB Broth, 600 µL of Tryptophan (a precursor to indole), 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.

To measure indigo production, we performed an extraction protocol on these overnights to extract indigo using DMSO and measured the absorbance on the plate reader. To convert the raw data, we made an indigo standard curve (shown below) and graphed the data below:

As shown in the graph above, there was a greater yield of indigo in the overnights that contained additional tryptophan. After we measured the overnights, we moved on to optimizing our system in cell-free. We set up cell-free reactions and graphed the results below.