Team:Stanford/Cell-Free Interlab

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ourTXTL | Accessible Cell-Free Collaboration

Content

Description

Design

Experiment

Results

ourTXTL | Collaborating to Bring Cell-Free to a Larger Audience

Description

Design

After encouragement from our mathematical model, we decided to design and order fragments that could express our dual-reporter promoter measurement standard in cell-free. We settled on using mRFP and Dronpa as our two fluorescent proteins as we included aptamers in our initial design and needed adequate separation in our excitation and emission spectra. However, we were unable to test our aptamer designs. We chose the J23101 promoter as our reference promoter given its strength in vivo and decided to measure the promoters J23102, J23113, J23150, and J23151 against it. Unfortunately, we did not acquire an mRFP construct driven by J23101 promoter, so we could not add relative strengths of our promoters. We collaborated with the University of La Verne’s iGEM team, who kindly sent us J2311, J2310, and I14018 to add to our list of promoters to characterize after one of our members met with them at their campus.

We planned on sending them our promoters to characterize in return but, as they did not have cell-free lysates to test them in, we sent them lyophilized extracts that we prepared (see later section) for them to test.

We wanted to validate our approach by taking the average mRFP fluorescence of each triplicate measurement and divide it by the average Dronpa fluorescence to normalize it. We expect to see the ratio of fluorescence to be relatively stable across time, which it happens to be. Interestingly, all of our promoters seem to have similar ratios, indicating similar relative strengths in our cell-free system.

Figure 1: Fluorescence ratios of dual promoter measurement standard. Cell-free was prepared as below and aliquoted into three 10 µL reactions.

Table 1. Samples were incubated at 30ºC in the plate reader and covered with 10 µL of mineral oil. Fluorescence was measured at 520 nm excitation and 610 nm emission to determine the amount of mRFP produced and 390 ex., 540 nm em. for Dronpa fluorescence.

Experiment

While we initially tested our fluorescence measurements in Arbor Bioscience’s myTXTL, we wanted to compare across different preparations of cell-free to observe if our relative strengths are consistent. Didovyk et al. (2017)’s protocol2 appealed to us given that the preparation lacks a sonicator and suggests that cell-free lysates prepared using this method could be lyophilized and stored at room temperature. We created a table (see QR code below) of necessary reagents to create this cell-free and implemented the freeze-thaw variant of the method (which we term ourTXTL) using the following procedure:

1. Grow 400 mL of 2x YT Media with ampicillin added. Add a tip of the pLyseR3 glycerol stock to the culture flask and shake at 37ºC until a 1:4 dilution of cells reaches an OD600 of ~0.3 (Mine was 0.228, which makes actual OD600 of culture 1.14).
2. Centrifuge the culture at 1800 x g for 15 minutes at room temperature.
3. Resuspend in 45 mL of S30A buffer. Vortex to mix the cells back in the culture.
4. Centrifuge the culture at 1800 x g for 15 minutes at room temperature.
5. Resuspend with S30A buffer with 2mM of DTT added. To determine the amount to add, measure the pellet and multiply the number of grams of cells harvested by 2 and add that many mL (this was technically challenging, so we assumed ~1 g of culture, and resuspended in 2 mL of S30A buffer plus 2 mM DTT). We put these cells in 2 2mL microcentrifuge tubes and used these tubes for the duration of the procedure.
6. Freeze the pellet at -80ºC. We left these overnight and continued work the next day.
7. Thaw the cells in a water bath at room temperature. You should be able to see the liquid move up and down as you turn the tube when it is fully melted.
8. Vortex for 3 minutes by taping the tube to the vortexer and turning it on (not using auto, but on).
9. Incubate in the 37ºC shaker for 45 minutes.
10. Repeat steps 8 and 9.
11. Put a centrifuge in the 4 degree room and let it cool. Centrifuge the pellet for 93 minutes at max speed (this is 17,000 x g for our microcentrifuge) in the 4 degree room. Pellet looked like this after centrifugation:

    

12. Pipet out the supernatant and add it to new microcentrifuge tubes. Spin again at max speed (17,000 x g) in the 4 degree room for 5 minutes (We saw no precipitate, so we did not move the supernatant to a new tube).
13. Freeze at -80ºC for future use.

Results

After confirming our lysates were functional (see figure below), we attempted to freeze-dry our E. coli lysates and, though attenuated, there was a clear distinction between lyophilized cells with GFP DNA added and those without. Because our measurement standard is based on relative activity, our lyophilized cell-free lysates could theoretically be used to support crowdsourced characterization of promoter strengths within cell-free.

Figure 2: Cell lysates translate GFP DNA. 16.9 µL of energy premix and mixed with 16.0 µL of cell-free lysate along with 1.0 µL of Murine RNAse inhibitor, 0.4 µL of 1 M IPTG, and 0.4 µL of concentrated T7 polymerase. 2 µL of 53.1 ng/µL GFP Plasmid DNA was added to this unlyophilized positive control. Lyophilized cell-free extract was prepared by adding 53.4 µL of energy premix to 48 µL of cell-free lysate and dehydrated using a commercial freeze dryer (Labconco). It was stored at room temperature for 5 days before being rehydrated with 70 µL of nuclease-free water. 1.0 µL of Murine RNAse inhibitor, 0.4 µL of 1 M IPTG, and 0.4 µL of concentrated T7 polymerase were added to 33.4 µL of the rehydrated lysate. Either 2 µL of 53.4 ng/µL GFP Plasmid DNA or 2 µL of nuclease-free water was added to our this mix. These were split into 10 µL aliquots and incubated for 15 hours at 29ºC and their fluorescence was observed. Note that replicates are denoted as mostly transparent colors and averages are denoted as solid lines.

Given our promising results creating and lyophilizing ourTXTL, our team plans on continuing to characterize performance of our extracts when freeze-dried and rehydrated and expand our collaboration to enable more teams to access cell-free. Additionally, we plan on using ourTXTL to validate our in vitro promoter strength measurement standard.