Biology subjects

Efromson, J. P.

Publications and source records attributed to Efromson, J. P..

3 recordsLinked to original sources

bioRxiv2020

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction in E. coli

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology. HighlightsO_LICharacterization of the impact of media on promoter activity C_LIO_LIIdentification of promoters robust to environmental variables C_LIO_LIIdentification of promoters whose expression scale from microtiter plates to bioreactors C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/920280v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@94f035org.highwire.dtl.DTLVardef@264edaorg.highwire.dtl.DTLVardef@102ee30org.highwire.dtl.DTLVardef@163d16e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

synthetic biology↗

bioRxiv2020

Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high throughput cultivation in microtiter plates and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion. HighlightsO_LIAutoinduction of both cell lysis and nucleotide hydrolysis C_LIO_LI>90 % lysis and DNA degradation C_LIO_LIStrains are stable to lysis in the absence of phosphate depletion. C_LI

synthetic biology↗

bioRxiv2019

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.

We report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of [~]30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/-0.7 g/L ([~]270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach [~] 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation. HighlightsO_LIStationary phase protein expression results in high titers. C_LIO_LIAutoinduction by phosphate depletion enables protein titers from 2-8 g/L. C_LIO_LIAutoinduction has been validated from 384 well plates to instrumented bioreactors. C_LI

synthetic biology↗