Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction

Rustad, Mark; Eastlund, Allen; Jardine, Paul J.; Noireaux, Vincent

doi:10.1093/synbio/ysy002

Cited by 93 publications

(64 citation statements)

References 39 publications

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“…S. aureus was grown using BHI media or BHI agar. LB agar (0.6%) was used for T4 plaque assays, made from 2 g tryptone, 1 g yeast, 1.2 g bacteriological agar (Thermo Fisher Scientific, Basingstoke, UK), 2 g NaCl per 200 ml (Rustad, Eastlund, Jardine, & Noireaux, ). BHI agar (0.7%) was used for phage K plaque assays, made from 7 g BHI media, 1.4 g bacteriological agar (Thermo Fisher Scientific) per 200 ml (O'Flaherty et al, ).…”

Section: Methodsmentioning

confidence: 99%

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial‐resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1–20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large‐scale bioreactors (up to 50 L) containing different bacteriophage–bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi‐liter large‐scale bioreactors is challenging in itself, but performing this for individual phage‐host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage–bacterial host bioprocesses (T4 – Escherichia coli; Phage K – Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake‐flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale‐up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550‐fold increase in bacteriophage yield and these improvements successfully translated to the large‐scale cultures.

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Section: Methodsmentioning

confidence: 99%

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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“…These cell-free approaches have facilitated the development of therapeutically useful natural products (Dudley et al, 2015;Maini et al, 2016) and biologics with non-standard amino acids (Martin et al, 2018) or chemical moieties otherwise challenging to synthesize (Jaroentomeechai et al, 2018). They have also been used to generate viable phage particles directly from purified phage DNA (Shin et al, 2012;Garamella et al, 2016;Rustad et al, 2018) and detect pathogens in emerging diagnostic applications (Pardee et al, 2014;Takahashi et al, 2018). Recently, cell-free transcription-translation (TXTL; Sun et al, 2013;Kwon & Jewett, 2015) has increasingly gained popularity as a prototyping platform for synthetic biology (Hodgman & Jewett, 2012;Takahashi et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

Yim

Johns

Park

et al. 2019

Molecular Systems Biology

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Cell‐free expression systems enable rapid prototyping of genetic programs in vitro . However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing ( DRAFTS ), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

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“…Since their first application in deciphering the genetic code [4,5], cell-free systems have been successfully applied for the bulk production of model [6][7][8][9] and therapeutic proteins [10][11][12][13][14][15]. Beyond just protein synthesis, though, CFE technologies have evolved more generally to enable complex and diverse functions, including prototyping cellular metabolism [16][17][18] and glycosylation [19][20][21], expressing minimal synthetic cells, virus-like particles, and bacteriophages [7,[22][23][24][25][26], portable on-demand manufacturing of pharmaceuticals [27,28], incorporation of nonstandard amino acids within proteins [29][30][31][32][33], prototyping of genetic circuitry [34][35][36], and sensing viral RNAs and small molecules through rapid, low-cost, and fielddeployable molecular diagnostics [37][38][39][40][41][42]. Most progress has occurred in CFE systems generated from Escherichia coli strains engineered for protein production, largely due to the bacterium's well-characterized genetics and metabolism [1].…”

Section: Introductionmentioning

confidence: 99%

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

Silverman¹,

Kelley‐Loughnane

Lucks³

et al. 2018

Preprint

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Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ 70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

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Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction

Cited by 93 publications

References 39 publications

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

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