Team:Bielefeld-CeBiTec/Results/CFPS

CFPS

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Motivation

Cell free protein synthesis has rarely appeared at iGEM in the recent years. This is opposed to quite many advantages (see here) CFPS has compared to cell-based approaches. We set out to establish CFPS protocols that can easily be performed by iGEM teams. We asked experts and gathered literature and information to be up to date in a research area that evolves at a grate pace.

Preliminary experiments

The extract of E. coli contains the molecular machinery that is needed for in vitro transcription and translation. For optimal yield of ribosomes, cell harvest has to be performed at mid- to late exponential growth phase. We therefore started to measure growth curves for various strains at 100 mL scale. The strains we investigated possess a genomic coded polymerase of bacteriophage T7. T7 polymerase is a monomer, very specific to its promoter and more stable than bacterial polymerases (Sousa and Mukherjee, 2003), which makes it perfectly suited for in vitro transcription.

Growth curves of the two E. coli strains ER2566 and KRX. Induction of T7-polymerase was faciliated by adding IPTG in the case of ER2566 and rhamnose in the case of KRX to the medium. For details see notebook.

So...

we observed that induction of T7-polymerase expression at OD₆₀₀ = 0.8-1.4 was not critical for growth kinetics. Regarding published data (Kwon and Jewett, 2015), we concluded that a cell harvest at an OD₆₀₀ = 3-4 would be optimal. At this stage of growth, the cells would have a highly active translation machinery, but they would still be far away from stationary phase.

What to do with the cells?

To generate crude cell extract, the bacteria have to be disrupted. This can be achieved by different methods, for example bead beating (Sun et al. 2013) or homogenizing with high pressure (Yang et al. 2012). However, we decided for sonification. A recent publication from Kwon and Jewett showed that it is fast, cheap, reliable, easy to perfom and that it works with small volumes (Kwon and Jewett, 2015) – which altogether is indicative for the use of this disruption method at iGEM competition.

The parameter with the greatest impact on crude cell extract quality is the sonifiers energy output (expressed in Joule per second) (Kwon and Jewett, 2015). Insufficient energy does not lyse cells efficiently, whereas too much energy inactivates proteins. Although we had access to a sonifier, we faced a problem: The device did not display its energy output. However, we tackled this problem by measuring how the temperature of a water sample changed as a function of sonification time. With an equation that takes all important parameters into account we were able to correlate the sonification time needed for a desired energy output.

Poor cells... any survivors?

We immediately wondered how effective our sonifier was when it comes to the disruption of E. coli. We cultivated, harvested and washed E. coli cells. Then we sonicated them in cycles of 10 seconds. We took aliquots and plated them onto LB plates to get a first idea about efficiency, and we observed a decrease in cell viability with increasing sonification cycles.

Sonification efficiency test. 10 µL aliquots were taken out of the 1.5 mL vessel containing the resuspended cell pellet and diluted in 990 µL. 20 µL of this samples were plated on LB. For details see notebook.

In the same manner we took aliquots after the subsequent centrifugation steps according to protocol, and viable cell numbers were even lower. During our project, we continued to investigate how many cells survived the process. In our fully optimized extract, a 100 µL aliquot of flash-freezed crude cell extract contained only 8 colony forming units. Our final biosensor device did not contain any living E. coli at all, like you can see here. Although 8 colony forming units in crude cell extract are still too many in purposes of biosafety issues, we conclude that our sonification is extremely efficient in decreasing viable cell numbers and therefore counteracts a potential energy depletion source in our final application.

Our reporter

An ideal reporter protein for in vitro protein synthesis is superfolder GFP, abbreviated sfGFP (Lentini et al. 2013). In our first experiments with selfmade E. coli cell extract, we used P_T7-sfGFP from the parts registry. In a Fluostar platereader, we measured a 10 fold increased fluorescence when compared to the negative control after 7.5 h. This was the first time we realized that we made it; in vitro sfGFP transcription and translation was possible with our extract!

Template optimization

But we were not yet content with the results, we were sure that a further optimization was possible. By literature screening, we designed a translation enhancing sequence (5'-untranslated region, 5'-UTR) and inserted it into P_T7-sfGFP, thereby creating P_T7-UTR-sfGFP, BBa_K1758102. Our assumption was that if translation was a bottleneck in our extract, this sequence would improve sfGFP production.

This was the case in vivo: We observed a faster production of sfGFP when the plasmid DNA contained 5'-UTR in front of sfGFP coding sequence. For mRFP normalization on OD₆₀₀ we faced the problem that mRFP emits fluorescence at 607 nm (Lentini et al. 2013). However, results for sfGFP and mRFP equally demonstrate the usefulness of 5'-UTR for protein production in general.

This was the case when 5'-UTR was employed in in vitro experiments: We observed a more than 3 fold increase in fluorescence. This clearly showed the importance of this enhancing element in CFPS, and further demonstrated that translation efficiency in vitro is a major issue for protein synthesis.

Importance of 5'-untranslated region (UTR) for in vitro protein synthesis. T7 refers to T7 promoter. sfGFP lysate refers to cell lysate won by sonification; cells were induced to produce sfGFP in vivo. Decreasing sfGFP lysate signal probably relies on evaporation.

Next steps

With these results, we had a good positive control plasmid for our following reactions. Nevertheless, we observed batch-to-batch variation in our extracts, a phenomenon also described in the literature (Takahashi et al. 2015). To exclude any batch-to-batch variation in following reactions, we set up a 5 L fermentation and harvested cell pellets to produce by far enough cell extract for the summer (details in the notebook).

We started using a Tecan platereader, constantly heating to 37 °C, to measure kinetics of sfGFP production. Measuring nearly every minute, we traced production of sfGFP in real time. Once again the effect of 5'-UTR was observed. In the same experiment, we determined that arsenic up to concentrations of 50 µg/L has little to no effect on sfGFP production in our cell extract. This means that CFPS is robust enough to serve as a basis for an arsenic detecting biosensor.

Tracing CFPS kinetics in Tecan platereader. RFU signals were normalized to first signal of sfGFP lysate. Purified GFP refers to the signal of a His-tagged GFP solution at a concentration of about 320 µg/mL.

Tracing CFPS kinetics in Tecan platereader. RFU signals were normalized to first signal of sfGFP lysate. "with 10 µg/L Ars" refers to a CFPS reaction with T7-UTR-sfGFP in which 10 µg/L of arsenic were present.

Tracing CFPS kinetics in Tecan platereader. RFU signals were normalized to first signal of sfGFP lysate. "with 50 µg/L Ars" refers to a CFPS reaction with T7-UTR-sfGFP in which 50 µg/L of arsenic were present.

Depending on E. coli strain and cell harvest, it can be necessary to perform a so called run-off reaction during cell extract preparation. We determined that 30 min of run-off reaction enable best performance for our extract.

With this, we established a robust cell free protein synthesis system for the production of sfGFP in our lab. The previous reactions were mostly performed in 364 well plates, so to test if our cell extract works on paper we went on with different papers as base for our reactions.

So what about this PRIA?