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myTXTL® – Cell Free Protein Expression

myTXTL® is a fast and easy-to-use solution for protein expression in vitro.

myTXTL® is a fast and easy-to-use solution for protein expression in vitro. Gene transcription (TX) and translation (TL) is executed in a single reaction tube by a highly efficient cell-free system that utilizes the endogenous TXTL machinery from E. coli. The all-in-one solution offers convenient one-step gene expression from a simple nucleotide template for various applications in synthetic biology and biomanufacturing.

The myTXTL® platform is based on the revolutionary TXTL technology developed by Vincent Noireaux, PhD at the University of Minnesota. The system employs the endogenous core RNA polymerase and primary sigma factor 70 (σ70) present in the E. coli cytoplasm. The technology has been well characterized and proven useful for a wide variety of applications such as high yield protein synthesis, prototyping of biomolecular networks, bacteriophage production and high-throughput protein expression analysis.

myTXTL® kits contain ready-to-use Master Mix comprised of E. coli cell extract, energy buffer and amino acids mix optimized for coupled TXTL to maximize experimental performance and consistency. Whether setting up gene circuits, de novo template DNA synthesis, or enhancing myTXTL® utilizing linear templates, the myTXTL® platform delivers compatible and convenient solutions for protein expression synthetic biology research.

Features & Benefits

All-in-one Solution
Simply mixing template DNA and ready-to-use myTXTL Master Mix.
Minimal investment
Only standard laboratory equipment required.
Fast processing
Save time by avoiding transformation, clone selection and cell lysis.
Multiple inputs
Start from plasmid, linear DNA or RNA templates.
Various expression systems
Compatible with E. coli-specific and T7 expression systems.
High-throughput screening
Process more samples within a single experiment.
  • Garamella J, Marshall R, Rustad M, Noireaux V. (2016) The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology. ACS Synth Biol 5, 344-355. DOI: 10.1021/acssynbio.5b00296
  • Caschera F and Noireaux V. (2014) Synthesis of 2.3 mg/mL of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie 99, 162-168. DOI: 10.1016/j.biochi.2013.11.025
  • Shin, J, Jardine P, and Noireaux V. (2012) Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol 1, 408-413. DOI: 10.1021/sb300049p

Unfortunately, this may lead to considerably decreased performance or even loss of function. To ensure highest kit performance, make sure to store Sigma 70 Master Mix at -80 °C and freeze as soon as possible after usage.

You`re advised to keep the number of freeze-thaw-cycles to a minimum. Nevertheless, our studies show that up to five freeze-thaw-cycles are acceptable without affecting protein production efficiency of the Sigma 70 Master Mix.

Yes. The myTXTL® Sigma 70 Master Mix contains tRNAs for seven codons rarely used in E. coli to prevent undesired translation stop.

As the myTXTL® platform completely relies on the endogenous transcription and translation machinery of E. coli making use of the core RNA polymerase and the primary sigma factor 70 (σ70), all genes should be cloned downstream of a σ70-specific promoter, e.g. the promoter found in P70a vectors. For a more general advice on how to construct a functional gene cassette, please refer to the myTXTL Manual.

Efficient in vitro protein production is highly dependent on the quality of the template DNA, which should be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery (e.g. EDTA, ethidium bromide, SDS, Cl- ions, ethanol). Preparation of plasmid DNA with standard commercial kits usually involves sample treatment with RNase, which may not be completely removed during downstream processing. Thus, we strongly recommend subjecting the prepared DNA to either a commercial PCR clean-up kit or standard phenol-chloroform extraction and ethanol precipitation. Ideally, template DNA is suspended in nuclease-free water. 
Please note, introducing Mg2+and K+ ions can compromise the kit performance, as they are extremely critical for transcription and translation, and are optimized in the systems.

deGFP is a N- and C-terminally truncated version of the reporter eGFP that is more translatable in cell-free systems. The excitation and emission spectra as well as fluorescence properties of deGFP and eGFP are identical.

Yes. Due to the manufacturing process, there might be a small pellet visible. It`s critical that you resuspend the Sigma 70 Master Mix completely before aliquoting it to set up your TXTL reaction(s).

Yes! That only requires the addition of a plasmid coding for T7 RNA polymerase under transcriptional control of a σ70-specific promoter, e.g. P70a-T7rnap. This plasmid – along with hundreds of others – is part of a Toolbox 2.0 Plasmid Collection and can be purchased here (LINK). The optimum concentration of P70a-T7rnap is usually between 0.1 nM and 1 nM. Higher concentration normally does not increase protein yield. The more important parameter for efficient protein expression is the concentration of the plasmid that encodes for your protein of interest downstream of the T7 promoter, which will be most likely in the range of 5-20 nM.

Yes, although it`s not optimized for linear DNA templates. Considerably enhanced protein yields can be achieved by supplementing the Sigma 70 Master Mix with our nuclease inhibitor GamS.

Due to the small reaction volume of 12 μL, it is very important to avoid condensation of water on lid of the reaction tube, as it considerably increases the concentration of myTXTL® components. This can lead to an unreproducible kit performance. In general, water facilitates a faster heat transfer than air and a water bath shows low temperature fluctuation, which should – combined with a closed environment with constant temperature surrounding the entire tube – lead to higher reproducibility and yield.

  • Component inactivation due to improper storage. Sigma 70 Master Mix must be stored at -80 °C and number of freeze-thaw cycles should be minimized.
  • Contamination of myTXTL® reaction with nuclease. To avoid nuclease contamination, wear gloves and use nuclease-free water, sterilized tips and tubes.
  • Please review the recommendations to set up a myTXTL® reaction in the myTXTL Manual.


Yes! Parameters that influence protein production efficiency are:

  • Gene cassette construction (promoter strength, position of affinity tag, TXTL elements)
  • Plasmid purity
  • Plasmid concentration
  • Incubation temperature and time
  • Presence of folding helpers, chaperones, oxidizing agents

and should therefore be evaluated for optimization. Please also see our recommendations on Template Design in the myTXTL Manual.


Consider if your recombinant protein requires co-factors like heavy metal ions or coenzymes to be functionally active. Those should be present during protein synthesis. Additionally, a low concentration of mild detergent (e.g. Triton-X-100, sodium dodecyl maltoside, or CHAPS) can be added to the reaction as well as molecular chaperones. Please note that the myTXTL® platform cannot introduce post-translational modifications like glycosylation or phosphorylation to your protein. Reducing the incubation temperature might help to prevent aggregation of the nascent polypeptide chain and to promote proper protein folding.

Unfortunately, not. However, studies have shown that supplementing cell-free systems with mixtures of reduced (GSH) and oxidized glutathione (GSSG), disulfide bond isomerase C (DsbC), protein disulfide isomerase (PDI) and/or chaperones (e.g. DnaK, DnaJ, GroEL, GroES) can promote the formation of disulfide bridges. In addition, pretreatment with iodoacetamide (IAM) to inactivate endogenous reductases which are present in the cell extract might also help (Review Article: Stech M & Kubick S, Antibodies 2015, 4, 12-33).

Batch-to-batch variation can be caused varying levels TXTL inhibitor contamination present in the plasmid solution. Please follow our recommendations on how prepare plasmid DNA as template for TXTL reactions which can be found in the myTXTL Manual.

Sample handling and storage is mainly determined by the stability of your molecule of interest (protein, DNA, RNA) and thus optimal conditions may need to be evaluated. But to ensure sample integrity, we would recommend to either process the myTXTL® reaction immediately after performing the incubation or store it at ≤ -20 °C.

Apart from standard biochemical methods like Coomassie-stained SDS-PAGE and western blot analysis, the great advantage of cell-free protein production is the open-system environment which allows the direct quantification and/or analysis of its functionality in an activity assay or the downstream processing via affinity purification (if an affinity tag is present). If you choose SDS-PAGE analysis, you can either take a small sample (1-3 µL) directly from your TXTL reaction, or – to reduce background signal – precipitate proteins with TCA/acetone or ammonium-acetate/methanol following a standard protocol.

Most importantly, the excitation and emission wavelength should match the fluorescence properties of deGFP/eGFP (e.g. λEm 488 nm, λEx 535 nm). Other reader settings such as reading mode, integration time and gain value should be chosen under consideration of high well-to-well fluorescence reading reproducibility.

 

Toolbox 2.0 Plasmid Collection


No. All our Toolbox 2.0 plasmids (except the positive control plasmid P70a-deGFP that comes with the myTXTL® kit) are meant for plasmid amplification in E. coli only. The degree of purity is NOT sufficient for efficient in vitro production. Please refer to the current myTXTL Manual for recommendations on preparation of plasmid templates for TXTL reactions.

Yes. For all plasmids containing the lambda phage promoter (P70a, P70b, P70c, P70d) it is extremely crucial to use E. coli KL740 as transformation strain. When cultivated below 30 °C, this strain over-expresses the lambda phage repressor protein Cl857, thus ensuring high transformation efficiency and plasmid stability. KL740 can be purchased from Coli Genetic Stock Center (Yale) [CGSC#: 4382]. For all other plasmids, a standard laboratory E. coli cloning strain like JM109 or DH5alpha is sufficient.

A standard protocol for chemical transformation usually produces E. coli cells with a sufficient competency for plasmid intake. We recommend following the procedure described in Sambrook et al. 1989.

Transformation efficiency extremely depends on the quality of the competent cells. Make sure that cells were immediately frozen after preparation and stored at ≤ 80 °C. Please also note that for some cells, transformation efficiency drops drastically over time. Additionally, we advise to use E. coli strain KL740 for amplification of any plasmids containing σ70-specific promoter like P70a.

All P70 promoters originate from the lambda phage promoter for the repressor Cro with its two operator sites and are specific to the E. coli sigma factor 70. They differ in strength (P70a > P70d > P70b > P70c) due to mutations that were introduced at -35 and/or -10 regions.

We do offer a comprehensive gene synthesis and cloning service (myDNA®) (LINK) that serves you with error-free DNA molecules at your convenience. Because we work with a modular part system, the cloning procedure will be straight forward with almost no limitations in combinatorial combinations at a very competitive pricing.

Yes. Please note, that every gene circuit should start with a σ70-specific promoter like P70a.

Fig 1. Workflow of in vitro protein expression with myTXTL

After template DNA – either circular or linear – has been added to the ready-to-use Sigma 70 Master Mix, in vitro protein expression starts immediately. The open-reaction environment allows easy manipulation of the system and straight-forward downstream processing of the recombinant protein.

 

 

Fig 2. Time-resolved protein expression in myTXTL

At certain time point of incubation, myTXTL reactions containing the positive control plasmid P70a-deGFP were analyzed by SDS-PAGE. A distinct band corresponding to the molecular weight of the expressed protein deGFP becomes visible over time.

 

 

Fig 3. Production of positive control deGFP using myTXTL

myTXTL Sigma 70 Master Mix with deGFP control plasmid before (A) and after (B) incubation at 29°C in a 1.5 mL reaction tube. (C) Fluorescence emitted by produced deGFP under UV light.

 

 

Fig 4. Effect of plasmid concentration on in vitro protein production.

deGFP expression is regulated by the interaction of the endogenous E. coli core RNA polymerase and the primary sigma factor 70 (σ70) with the σ70-specific promoter P70a.

 

 

Fig 5. Protein synthesis in myTXTL using the T7 expression system.

deGFP expression under the control of the bacteriophage T7 promoter (PT7) is facilitated by initial expression of T7 RNA polymerase from an additional plasmid.

Ordering Information

Catalog # Description Quantity Price Contact Us
507024 myTXTL Sigma 70 Master Mix Kit 24 Rxns $150 Order Now
507096 myTXTL Sigma 70 Master Mix Kit 96 Rxns $500 Order Now
501024 GamS Purified Nuclease Inhibitor Protein 24 Rxns $65 Order Now
501096 GamS Purified Nuclease Inhibitor Protein 96 Rxns $150 Order Now
50xxxx Toolbox 2.0 Plasmid 1 µg $50 Order Now

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