A main part of our project is the expression of proteins that will produce an observable signal. We can achieve such results by either in vivo transcription and translation in bacteria or in vitro utilizing cell lysates or recombinant systems. Since Genetically Modified Organisms (GMOs) are very difficult to use outside the lab in Europe, but we want our diagnostic test to be field-based, we opted to use a cell-free expression system.
Cell lysates, even though they can function very efficiently, are more complex both during production and implementation, so our final solution was to use a recombinant system.
The most popular system, which is used by molecular biologists all over the world, was introduced by Shimizu et al. in 2001 and is called PURE (Protein synthesis Using Recombinant Elements) and among others, it requires a solution of 36 different purified proteins. However, the production of PURE is quite costly, even for big corporations that are able to produce it in large quantities and can afford automated systems, and also very arduous, especially for small research teams.
To cope with this problem, we decided to produce our own PURE system based on an innovative new method that was developed at Maerkl Lab, EPFL (Lavickova et al., 2019). Here, instead of culturing the 36 protein producing strains in different flasks and then performing as many purifications, we co-culture them in one flask, hence the name “OnePot”, so only one purification step is required. With this method we can achieve a 14-fold decrease in the cost of each cell-free reaction (based on commercially available products) while we still get high yields on the produced proteins. In fact, implementing the OnePot method for producing the PURE system, instead of the commercial price of 1.36 USD/μl, we end up with a cost of 0.09 USD/μl.
A great current application of using the PURE cell-free system for detecting deadly diseases is Pardee et al., 2016. It describes a new cell-free, paper-based sensor for detecting the Zika virus RNA genome. Their freeze-dried biomolecular platform resolves important practical limitations to the deployment of point-of-care diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises.
In order for a gene to be expressed, we need a transcription-translation system. That includes, apart from the Ribosomes, some enzymes which are involved in different steps of protein synthesis.
One of the most important components of the PURE system is the T7 RNA Polymerase (T7 RNAP) which allows us to transcribe DNA sequences that have a T7 promoter to mRNA that will then be translated.
We may then separate protein synthesis in three steps: Initiation, Elongation and Termination. For Initiation we need to include 3 initiation factors (IF1, IF2 and IF3). Elongation requires 20 different Aminoacyl tRNA synthetases alongside with 3 elongation factors (EF-G,EF-Tu and EF-Ts), and finally 3 release factors (RF1,RF2 and RF3) and the Ribosome recycling factor (RRF) will conduct the Termination step.
In addition to the above-mentioned proteins, we still need to include 4 more enzymes that will catalyze the energy reactions and contribute to energy recycling, as well as the Inorganic pyrophosphatase (PPiase) that is responsible for regulating the secondary structure of the expressed proteins. A list of the 36 proteins needed for the PURE system can be found in the Annex section.
The proteins, apart from their functional domain, also include a histidine tag (His tag) that will allow for purification. The ribosomes, which are the main components for translation, are purified, on the other hand, utilizing the methods of hydrophobic interaction chromatography (HIC) and sucrose cushion buffer ultracentrifugation. Although this technique is simple to use and does not require genetically modified microorganisms (the ribosomes are collected from E.coli strain A19 bacteria), it might not be an appropriate solution for smaller labs due to the high cost of the equipment that is required. An alternative to this method is purification of his-tagged ribosomes or using a commercially available solution.
Last but not least, the Energy solution consists of amino acids and polyamines, tRNA, NTPs,salts, acids and necessary buffers. The Energy solution occupies 40% of each reaction and can be easily produced using commercially available materials.
In this section we will describe the methods that we implemented during the production of the OnePot PURE cell-free system. You can find the protocols we used here.
Buffers are used in almost all the steps of Protein and Ribosome purification. Some of the most important components are the following:
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was used in all the buffers as it prevents changes of pH despite any fluctuations in carbon dioxide concentration.
Imidazole competes with the his-tag for binding to the metal-charged resin (Ni-NTA in our case), so we can use it for both washing and elution. After the proteins are attached to the resin, we introduce low concentration of imidazole which interferes with the weakly bounded proteins, eluting them in the process. In the same manner, to elute the strongly bounded proteins we introduce a solution with high imidazole concentration.
Sucrose is the main component of the high salt sucrose cushion buffer that helps us isolate the proteins through ultracentrifugation.
Finally, β-Mercaptoethanol is used in all buffers to inhibit the oxidation of free sulfhydryl residues, and hence maintain protein activity. However, as its half-life is quite short, we need to add it to the buffer just before each use, unlike all the other components.
All the buffers can be stored at 4°C.
The preparation of the energy solution is a straightforward procedure, explained in more detail in our protocol here.
All we need to do is add the components that are responsible for the translation (tRNA, Amino Acids), energy production and transformation (NTPs) as well as the necessary salts, acids and buffers that affect the reactions that need to take place.
The protein purification is based on the technique of gravity flow affinity chromatography.
We start by culturing overnight the 36 different protein producing E.coli strains need in a deep well plate, except for the elongation factor EF-Tu, which is the most important component in this protein solution and has to be cultured in a tube as we need a lot more volume than a deep well allows us to put.
Then we move on to what makes this method so unique, which is the coculture in a single flask. The overnight cultures inoculate 500ml of LB medium (the volume of the EF-Tu culture that is used to inoculate the new solution is 30 times more than any other protein) that is then incubated before we add IPTG. This component will induce transcription of the desired proteins as they are regulated by a Lac operon.
The next step is to harvest and lyse the cells using sonication. The lysate, after removal of the unwanted debris, is mixed with the Ni-NTA resin and left in the fridge until the His-tags are bound to the resin. This step is the one that affects our final cost the most, as in the conventional PURE system preparation method we need 36 different column-based purifications in parallel. In addition, the resin can be easily regenerated and used multiple times.
Using different concentrations of Imidazole, as stated in the previous section, we manage to wash all the undesired proteins and finally elute the ones we need. Finally, the buffer exchange is achieved using an Amicon filter rather than dialysis. Even though dialysis is a simple method, it requires a greater amount of time, while filtering just takes a couple of hours.
The sample is then diluted with a glycerol-based buffer to allow for long-term storage. After that, it is concentrated to 12.25 mg protein per ml and is stored in a -80°C freezer and can be used without any further preparation.
Ribosome Purification with HIC:
Ribose purification is based on hydrophobic interaction chromatography (HIC).
We culture E.coli strain A19 bacteria in two steps, a small culture overnight and a larger one the next day, before we harvest the cells by centrifugation. After we lyse the cells, a two-step centrifugation is needed to remove the unwanted debris. First we centrifuge the lysate and then we repeat the procedure with the addition of a high-salt suspension buffer, before passing the solution through a syringe membrane filter.
The HIC purification takes place in an Akta Purifier using HiTrap Butyl HP columns. To identify the eluted fractions containing ribosomes, we check the absorbance peak at 280nm. Once again, we can regenerate the columns and reuse them for future purifications.
To harvest the ribosomes, we implement sucrose cushion buffer ultracentrifugation for 16 hours. This part is tricky, as after removing the supernatant the ribosome pellet might not be visible. We then resuspend with the appropriate buffer (according to the protocol) and concentrate it using a 3kDa Amicon Ultra filter by centrifugation. The last step is to calculate the concentration by measuring its absorbance at 260nm (since the ribosome solution is too costly, it is advised to dilute it to 1:100 ratio).
The ideal concentration is 10μΜ and can be achieved through dilution. We then we store it at -80°C.
Ribosome Purification with His-tag:
As mentioned before, Ribosome Purification with His-tags can be an alternative choice. (Ederth et al., 2009) designed a new strain of Escherichia coli (JE28) that produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Then, the purification is achieved using affinity chromatography.
The produced ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation have the advantage of not requiring specialized equipment, while maintaining high levels of efficiency.
PURE cell-free system assembly:
We can express any protein sequence equipped with a T7 promoter.
Each 5μl reaction needs 2μl of Energy Solution, 0.65μl of Protein Solution, 0.9μl of Ribosomes and the DNA template whose final concentration in the solution should be ideally 5nM. We add water to achieve a final volume of 5μl.
Although proteins can be expressed in such small volumes, we advise the users to perform 10μl reactions to minimize errors due to inaccurate pipetting.
A tutorial of the methods we used can also be found in the video below, as well as on the Education page
Ribosome purification was not included due to logistical issues, as the experiment took place in a specialized facility on our campus and having additional people to film the process would cause capacity problems.
We tested the functionality of the OnePot PURE that we produced using two different methods.
First, we performed an SPS-PAGE to compare our protein production with the one that appears on the reference paper we used. Since the author of the OnePot PURE paper is one of our advisors, it was convenient for us to replicate the same conditions. In the following image you can see a scan of the SDS gel for two different batches of Protein solution we produced, with two replicas each (right side of the gel):
As is clearly shown, all the bands are aligned, so we can conclude that the purification was successful. However, this result does not guarantee proper functionality of the protein solution.
Finally, we tested the ability of our OnePot PURE system to express super folding GFP.
We initiated 10μl reactions using our own Protein Solution, Energy Solution and Ribosome Solution, while altering the concentration of the DNA template. The goal of this experiment was to determine the optimal concentration of DNA in each reaction. In addition, we included similar reactions of PURExpress from NEB to compare our system to the commercially available one.
In the following graphs we show the Fluorecein concentration of the samples, measured at an excitation wavelength of 535 nm, using 2.5, 5 and 10nM of DNA template.
As we can see, OnePot PURE has an even higher expression of sfGFP than PURExpress*. The initial hypothesis, that 5nM of DNA is the optimal concentration, is also confirmed. We still get slightly higher fluorescence at 10nM but the difference is insignificant when taking into account that the concentration of DNA in our sample doubled.
*Because of the extreme difference between the output of OnePot and PURExpress, we repeated the experiments to rule out the effect of a human error using a different batch of PURExpress, but we got the exact same results.
Lavickova, Barbora, and Sebastian J. Maerkl. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell - Free System. preprint, Synthetic Biology, 18 Sept. 2018. DOI.org (Crossref), doi:10.1101/420570.
Cell-free translation reconstituted with purified components. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Nat Biotechnol. 2001 Aug;19(8):751-5. doi: 10.1038/90802. 10.1038/90802 PubMed 11479568
Ederth, Josefine, et al. “A Single-Step Method for Purification of Active His-Tagged Ribosomes from a Genetically Engineered Escherichia Coli.” Nucleic Acids Research, vol. 37, no. 2, Feb. 2009, p. e15. PubMed Central, doi:10.1093/nar/gkn992
Pardee K, Cell 165, 1255–1266, May 19, 2016. doi: 10.1016/j.cell.2016.04.059