MYtxtl® an E. coli Based Cell-Free Transcription-Translation (TXTL) Kit for Bio-Manufacturing

At present, hybrid bacteriophage-E. coli TXTL systems are popular and widely applied cell-free TXTL platforms in synthetic biology and bioengineering applications. By combining a strong and robust bacteriophage RNA polymerase (often from T7) for gene transcription with a cytoplasmic E. coli cell extract providing the translation machinery, this technology has proven useful for protein design and engineering, especially in high-throughput setups. Their major disadvantage is the relatively small repertoire of transcriptional regulators, thus considerably limiting the complexity of in vitrodynamical systems that can be constructed, such as gene circuits, for synthetic biology applications.

 Our MYtxtl^® kit – entirely based on the endogenous TXTL machinery of E. coli – allows the implementation of a plethora of transcriptional activation units, thus providing a unique and unrivaled degree of regulatory freedom for dynamical systems in gene circuitries. We make use of the primary E. coli transcription machinery consisting of the core RNA polymerase and sigma factor 70 (s⁷⁰) as basic transcriptional regulator and complement it with other stable or degradable endogenous transcription factors for setting up multi-stage activation cascades. In addition, gene expression under the control of the bacteriophage T7 promoter/operator system is still feasible by using a two-plasmid system: one for the expression of T7 RNA polymerase under transcriptional control of the E. coli P₇₀promoter, the other comprising the gene of interest downstream of the T7 promoter.

 Another promising, high potential field of application for cell-free TXTL systems is the large-scale protein synthesis (bio-manufacturing) for e.g. the production of biopharmaceuticals. Currently, major hurdles to enter this market are that (i) existing methods for cell extract preparation are developed for standard shake flask cultures only providing merely insufficient amounts of bacterial cell pellet as well as bear the risk of batch-batch variation and (ii) protein expression in > 20 µL TXTL reaction volume is limited most likely due to poor and inconsistent supply of oxygen which decelerates the TXTL machinery.

 To overcome the first limitation, we investigated the preparation of E. colicell extracts using a large fed-batch fermenter by permanently adjusting pH, temperature and supply of nutrients. This approach yielded in considerably higher cell densities during log-phase cell growth as compared to flask-production, giving TXTL reactions with proportionate protein production ability.

 Secondly, we developed scaled-up TXTL reactions by evaluating the effect of several oxygenation systems and incubation methods on their protein production efficiency. Thereby, we demonstrated that high-yield production of correctly folded and biochemically active protein in TXTL milliliter-scale reactions is feasible.

Thus, by combining the regulatory flexibility of the E. coli TXTL machinery with effective oxygenation methods, we developed a cell-free TXTL systems applicable for both synthetic biology and high-mass protein production.