I accidentally stored the myTXTL® Sigma 70 Master Mix at room temperature/at 4 °C. Can I still use it?
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.
Do I have to expect loss of production efficiency due to freezing and thawing of the myTXTL® Sigma 70 Master Mix?
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.
Is it possible to express genes of eukaryotic origin that were not codon optimized for E. coli?
Yes. The myTXTL® Sigma 70 Master Mix contains tRNAs for seven codons rarely used in E. coli to prevent undesired translation stop.
What plasmid design parameters should I consider when using the myTXTL® Sigma 70 Master Mix system?
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.
Do you have recommendations on how to prepare the plasmid template for the myTXTL® Sigma 70 Master Mix reaction?
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.
What is the difference between enhanced green fluorescent protein (eGFP) and deGFP?
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.
After thawing and centrifugation of the myTXTL® Sigma 70 Master Mix, there is a small pellet at the tube bottom. Is that normal?
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).
My protein is under transcriptional control of the T7 promoter system. Can I use it for protein expression in the myTXTL® Sigma 70 Master Mix kit?
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.
Does the myTXTL® Sigma 70 Master Mix also work with linear DNA templates?
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.
What device do you recommend for incubating myTXTL® reactions (incubator, thermoblock or water bath)?
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.
Why am I unable to synthesize the positive control protein deGFP from P70a-deGFP plasmid in myTXTL®?
- 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.
I was successful in synthesizing the control protein deGFP, but my protein of interest is not or only present in low yield in the myTXTL® system (using a plasmid DNA template). Is there anything I can do?
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.
What should I do if my protein is not functionally active and/or forms aggregates?
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.
Does the myTXTL® system promote the formation of disulfide bonds?
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).
Why do I observe a different protein yield depending on the plasmid DNA batch?
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.
Can I freeze a myTXTL® reaction after the incubation has been completed?
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.
What are options to analyze my samples after running the TXTL reaction when using the myTXTL® system for protein production?
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.
Do you have any recommendations regarding the settings of the fluorescence reader for deGFP/eGFP quantification?
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.
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.
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.
deGFP expression under the control of the bacteriophage T7 promoter (PT7) is facilitated by initial expression of T7 RNA polymerase from an additional plasmid.