T7Max transcription system

Abstract

Background: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.

Results: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.

Conclusions: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Keywords: cell-free protein expression; in vitro transcription; in vitro translation; synthetic cells.

Fig. 1

Testing different promoters in in-vitro transcription. a transcription of the RNA broccoli aptamer from linear dsDNA templates under different promoters. The gels are stained with DFHBI1T. b quantification of DFHBI1T stained gels. Y axis is the unitless relative brightness of the broccoli RNA band. For the gels shown on panel a and c we used sample of purified Broccoli aptamer as size standard. Original uncropped gel images are shown on figures S1 and S2. c quantification of the same transcription gel as in a, stained with Sybr stain. d quantification of the Sybr stained gel. The Y axis is unitless relative brightness of the aptamer RNA band. e time course of transcription from linear dsDNA aptamer templates with different promoters, one example trace for each experiment. The legend applies to panels e and f. f: end point fluorescence of RNA aptamer for 3 replicates for transcriptions showed on panel e, fluorescence measured at excitation 488nm and emission 507 nm; error bars are standard deviation

Fig. 2

Cell-free TxTl of GFP from dsDNA linear template with different promoters. a cell-free TxTl synthesis of eGFP, with two top candidate promoters, end point fluorescence measured after 8-hour reactions. b: RT-qPCR measurement of mRNA abundance in TxTl GFP translation of classic T7 promoter, new T7 Max promoter, and no template control sample. Samples were collected after an 8 -hour TxTl reaction. c: cell-free TxTl synthesis of GFP, T7 promoter (green bars) and T7Max promoter (blue bars), in house -made bacterial TxTl, with different ways of protecting linear DNA templates, and with commercially available myTXTL kit; end point fluorescence measured after 8 -hour reactions. For panels a, b and c each sample in triplicate, error bars are standard deviation. d example of Western Blot analysis of GFP translation, 8 -hour end point translation from linear dsDNA template in home-made TXTL without DNA protection reagents (samples represent conditions showed on panel c marked with red star). All TxTl reactions were incubated at 30°C

Fig. 3

Cell-free TxTl of GFP from dsDNA circular plasmid template with different promoters. a time course expression of GFP under the classic T7 vs T7Max promoter. b Western Blot analysis of expression of GFP. c RT qPCR cycle (Cq) value quantifying abundance of GFP mRNA. Full, uncropped image of this gel is on Figure S6. d quantification of Western Blots of GFP expression, expressed as unitless relative brightness value. All samples in triplicate, error bars represent S.E.M. Protein product was measured by endpoint measurements after an 8 -hour reaction. All TxTl reactions were incubated at 30°C

Fig. 4

T7Max performance characterization. Translation of GFP protein from circular plasmid template was measured at different temperatures and with different T7 RNA polymerase concentration. All green bars: T7 promoter, all blue bars: T7Max promoter. a expression of GFP measured after an 8-hour reaction at different temperatures. b RT qPCR measuring abundance of GFP mRNA in samples from panel a. c mRNA abundance measured at different times during the TxTl reaction at 30°C. d expression of GFP measured after an 8 hour reaction with different concentration of T7 RNA polymerase, TxTl reaction at 30°C. The percentage numbers above bars show fluorescence relative to the value at 2.5μM T7 RNAP for each promoter. All samples in triplicate, error bars represent standard deviation. The concentration of T7 RNAP was varied by adding different amounts of 25μM stock of overexpressed, purified T7 RNA polymerase, stock in 50% glycerol

Fig. 5

Performance of T7Max vs T7 promoter in different template lengths. All green bars: T7 promoter, all blue bars: T7Max promoter. Circular plasmid DNA template expression of proteins with different length of the open reading frame, from 1650 base pairs to 30 base pairs. Each graph shows protein product quantification and corresponding RT qPCR cycle (Cq) value quantifying abundance of mRNA for each protein. All samples in triplicate, error bars represent S.E.M. Protein product was measured by end point measurements after an 8- hour reaction. Luminescence with appropriate luciferase product was used on panels a, d, f. Quantification of appropriate size Western Blot band, expressed as unitless relative brightness value, was used on panels b, e, g and h. Original uncropped gels are on Figure S7. Fluorescence with the arsenic ligand was measured on panel i

Fig. 6

Synthetic minimal cells expressing GFP protein. Microscope images showing liposomes encapsulating plasmid encoding GFP under T7 (panels a and c) and T7Max (panels b and d) promoters. Panels a and b: 0.1mM lipid concentration, green (GFP) and red (rhodamine membrane dye) channels overlayed. Panels c and d: bright field showing density of liposomes at 10mM lipid. Scale bar is 5μm. e and f: quantification of 5 images taken from different fields of view in samples at 0.1mM lipid (panel e) and at 10mM lipid (panel f). Error bars represent standard deviation. The value is ratio of total fluorescence in green channel to total fluorescence in red channel

Fig. 7

Performance of T7Max in different systems. a Cell-free translation reactions based on different organisms. GFP plasmids were prepared for each specific commercial cell-free expression system (except E. Coli, which used the same plasmids as tested earlier, and in house made cell-free expression system). Fluorescence of GFP protein was measured after each reaction, and raw fluorescence was normalized so that classic T7 promoter fluorescence was assigned value 100, and T7Max sample fluorescence was scaled proportionally. All samples are in triplicate, error bars represent standard error. The raw fluorescence data for all normalized data points are on Figure S8, and the method for calculation of error bars (error propagation) is described in Materials and Methods section “Promoter comparison using different extracts”. b Apta-Nucleic Acid Sequence Based Amplification reaction detecting E. Coli gene, aggR. Reactions are identical except for the incorporation of T7Max vs classic T7 promoter. Fluorescence of the broccoli aptamer was measured every 2.5 minutes, excitation: 488 nm and emission: 507 nm, with PMT sensitivity set to Medium for all readouts. All samples were performed in triplicate, and traces represent the average