Overview

The myTXTL Toolbox 2.0 Plasmid Collection was designed and intensively studied by Vincent Noireaux, PhD at the University of Minnesota, and co-workers. It contains more than 100 plasmids with various promoters and open reading frames (ORFs) to investigate gene regulation and molecule turnover. Available ORFs include a wide selection of transcription factors, TXTL modulators, and fluorescent reporter proteins to build multi-stage gene circuits.

A gene circuit executed in any myTXTL kit is required to start with a σ70-specific promoter, as gene expression in myTXTL relies entirely on the endogenous TXTL machinery of E. coli. Its modular and pre-designed format makes it ideal for training students and demonstrating basic concepts of synthetic biology.

Important: Toolbox 2.0 plasmids are intended for propagation purposes only. To make a plasmid directly suitable for in vitro expression in myTXTL, follow the recommended two-step procedure to generate high-purity plasmid samples. For propagation of P70a-vector, please read the Tech Note, Preparation of chemo-competent KL740 cells for amplification of P70a vectors.
 
NOTE: These products will no longer be available after August 5, 2024. Click here for important information about upcoming myTXTL product line changes.

Applications

The combination of synthetic gene networks with in vitro protein production technology opens new innovative fields of application in medicine and biotechnology, including:

  • Environmental sensors
  • Controls for biomanufacturing of biofuels
  • Stem cell medicine
  • Gene therapy (CRISPR)
  • Manufacturing of functional materials

Cell-free platforms help to overcome limitations occurring in living host organisms, which are typically tied to the biochemical cross-talk between the host cell and the artificial gene circuit

and a consequence of the potentially cytotoxic effect of the exogenous gene network on the living cell on the one hand, and the availability of resources for transcription and translation, DNA replication and metabolites on the other. As those resources are dependent on the cell density, growth rate and cultivation conditions, deploying cell-free systems for gene circuit testing creates a more controlled environment, increasing reproducibility and robustness of the circuit behavior and output.

Performance

The myTXTL Toolbox 2.0 Plasmid Collection serves as a comprehensive source to build a variety of gene circuits executable in the myTXTL system. The examples below highlight selected features and capabilities. For more inspiration, visit our publication page.

Utilizing a two-gene transcriptional cascade for T7 promoter expression

To facilitate T7 promoter regulated gene expression in myTXTL, T7 RNA polymerase is co-expressed from a helper plasmid (P70a-T7rnap) encoding a Sigma70-specific promoter compatible with myTXTL Master Mixes. T7 RNA polymerase then facilitates gene expression on the T7 promoter DNA template (here: T7p14-deGFP plasmid). Constitutive and inducible T7 promoter versions are acceptable.

Execution of biologic logic gates in myTXTL

In synthetic biology, logic gates research is aimed at the development of decision-making gene networks for environmental and medical applications, in which biological gates act as sensors, detection units and drug vehicles. In this AND gate circuit, deGFP expression requires both phosphorylation of Nitrogen regulator protein C (NtrC) as well as presence of transcription factor Sigma54.

Rapid prototyping of synthetic gene networks

Engineering of synthetic gene networks involves executing an iterative design-build-test cycle which is accelerated by cell-free expression technology like myTXTL. By utilizing the native E. coli TXTL machinery, myTXTL gives access to an extraordinarily large repertoire of genetic building blocks. Here, a multi-stage cascade composed of a logic AND gate and bacterial transcription factors is producing a fluorescent output. Other fluorescent proteins are part of the myTXTL Toolbox 2.0 Plasmid Collection.

Compartmentalization of cell-free reaction

The encapsulation of myTXTL reactions into large phospholipid vesicles (liposomes) is an example of how artificial cells hosting cellular functions and complexity as advanced as in nature are constructed. Incorporation of transmembrane proteins provides more sensing options and facilitates cellular communication. Other approaches of compartmentalization such as emulsions, liquid-liquid phase separation, hydrogels and polymer-containing membrane compartments can also be applied.

FAQs

Transformation efficiency depends on the quality of the competent cells. Make sure that cells were immediately frozen after preparation and stored at ≤ 80 °C. Please also note that for some cells, transformation efficiency drops drastically over time. Additionally, we advise to use E. coli strain KL740 for amplification of any plasmids containing σ70-specific promoter like P70a.

No. All our Toolbox 2.0 plasmids (except the positive control plasmid P70a-deGFP that comes with the myTXTL kit are meant for plasmid amplification in E. coli only. The degree of purity is NOT sufficient for efficient in vitro production. Please refer to the current myTXTL handbook for recommendations on preparation of plasmid templates for myTXTL reactions.

All P70 promoters originate from the lambda phage promoter for the repressor Cro with its two operator sites and are specific to the E. coli sigma factor 70. They differ in strength (P70a > P70d > P70b > P70c) due to mutations that were introduced at -35 and/or -10 regions.

Yes, please see examples in the Publications section; filter for myTXTL. Please note, that every gene circuit should start with a σ70-specific promoter like P70a.

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 master mix.

Yes. For all plasmids containing the lambda phage promoter (P70a, P70b, P70c, P70d) it is extremely crucial to use E. coli KL740 as the transformation strain. When cultivated below 30°C, this strain over-expresses the lambda phage repressor protein Cl857 that represses P70 promoters, thus ensuring high transformation efficiency and plasmid stability. KL740 can be purchased from E. coli Genetic Stock Center (Yale) [CGSC#: 4382] or from Daicel Arbor. For all other plasmids, a standard laboratory E. coli cloning strain like JM109 or DH5alpha is sufficient.

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Ordering Information
Cat#
Qty
Price
pTXTL-P19a-S24
P19a-S24
502005
$80
pTXTL-P19a-S28
P19a-S28
502007
$80
pTXTL-P19a-S28-ssrA
P19a-S28-ssrA
502008
$80
pTXTL-P19a-S38
P19a-S38
502009
$80
pTXTL-P19a-deCFP
P19a-deCFP
502002
$80
pTXTL-P19a-deGFP
P19a-deGFP
502001
$80
pTXTL-P19a-deYFP
P19a-deYFP
502003
$80
pTXTL-P19a-ntrC
P19a-ntrC
502004
$80
pTXTL-P19a-ompA-S24
P19a-ompA-S24
502006
$80
pTXTL-P24a-S28
P24a-S28
502011
$80
pTXTL-P24a-S28-ssrA
P24a-S28-ssrA
502013
$80
pTXTL-P24a-S38
P24a-S38
502014
$80
pTXTL-P24a-deGFP
P24a-deGFP
502010
$80
pTXTL-P24a-ompA-S38
P24a-ompA-S38
502012
$80
pTXTL-P28a-aH
P28a-aH
502015
$80
pTXTL-P28a-aH-eGFP
P28a-aH-eGFP
502018
$80
pTXTL-P28a-cI-ssrA
P28a-cI-ssrA
502017
$80
pTXTL-P28a-S19
P28a-S19
502031
$80
pTXTL-P28a-S19-ssA
P28a-S19-ssA
502032
$80
pTXTL-P28a-S24-ssrA
P28a-S24-ssrA
502033
$80
pTXTL-P28a-S38
P28a-S38
502035
$80
pTXTL-P28a-S54
P28a-S54
502036
$80
pTXTL-P28a-T7rnap
pTXTL-P28a-T7rnap
502118
$80
pTXTL-P28a-deGFP
P28a-deGFP
502019
$80
pTXTL-P28a-deGFP-ssrA
P28a-deGFP-ssrA
502020
$80
pTXTL-P28a-deYFP
P28a-deYFP
502021
$80
pTXTL-P28a-flgM
P28a-flgM
502022
$80
pTXTL-P28a-flgM-ssrA
P28a-flgM-ssrA
502023
$80
pTXTL-P28a-mApple
P28a-mApple
502025
$80
pTXTL-P28a-mazE-ssrA
P28a-mazE-ssrA
502027
$80
pTXTL-P28a-mreC
P28a-mreC
502029
$80
pTXTL-P28a-mreB
P28a-mreB
502028
$80
pTXTL-P28a-ntrC
P28a-ntrC
502030
$80
pTXTL-P28a-ompA-S24-ssrA
P28a-ompA-S24-ssrA
502034
$80
pTXTL-P28a-tetO1-deGFP-ssrA
pTXTL-P28a-tetO1-deGFP-ssrA
502132
$80
pTXTL-P28a-tetR
P28a-tetR
502037
$80
pTXTL-P28a-tetR-ssrA
pTXTL-P28a-tetR-ssrA
502131
$80
pTXTL-P28a-venus-mreB
P28a-venus-mreB
502038
$80
pTXTL-P28a-venus-mreB-18L
P28a-venus-mreB-18L
502039
$80
pTXTL-P32a-deGFP
P32a-deGFP
502040
$80
pTXTL-P38a-S19
P38a-S19
502043
$80
pTXTL-P38a-S19-ssrA
P38a-S19-ssrA
502044
$80
pTXTL-P38a-S28
P38a-S28
502045
$80
pTXTL-P38a-S28-ssrA
P38a-S28-ssrA
502046
$80
pTXTL-P38a-S54
P38a-S54
502047
$80
pTXTL-P38a-deGFP
P38a-deGFP
502041
$80
pTXTL-P38a-ntrC
P38a-ntrC
502042
$80
pTXTL-P54a-S19
P54a-S19
502049
$80
pTXTL-P54a-S24
P54a-S24
502050
$80
pTXTL-P54a-S28
P54a-S28
502051
$80
pTXTL-P54a-S38
P54a-S38
502052
$80
pTXTL-P54a-deGFP
P54a-deGFP
502048
$80
pTXTL-P70a-broccoli
pTXTL-P70a-broccoli
502119
$80
pTXTL-P70a-cI
pTXTL-P70a-cI
502120
$80
pTXTL-P70a-cI-ssrA
P70a-cI-ssrA
502054
$80
pTXTL-P70a-clpXP
P70a-clpXP
502053
$80
pTXTL-P70a-deCFP
P70a-deCFP
502055
$80
pTXTL-P70a-S19
P70a-S19
502067
$80
pTXTL-P70a-S19-ssrA
P70a-S19-ssrA
502068
$80
pTXTL-P70a-S24
P70a-S24
502069
$80
pTXTL-P70a-S28
P70a-S28
502072
$80
pTXTL-P70a-S28-ssrA
P70a-S28-ssrA
502073
$80
pTXTL-P70a-S28-ybaQ
P70a-S28-ybaQ
502074
$80
pTXTL-P70a-dTomato
P70a-dTomato
502060
$80
pTXTL-P70a-deGFP
P70a-deGFP
502056
$80
pTXTL-P70a-deGFP-ssrA
P70a-deGFP-ssrA
502057
$80
pTXTL-P70a-deYFP
P70a-deYFP
502058
$80
pTXTL-P70a-dmVenus
P70a-dmVenus
502059
$80
pTXTL-P70a-luc
pTXTL-P70a-luc
502121
$80
pTXTL-P70a-mApple
P70a-mApple
502061
$80
pTXTL-P70a-mGapt-deGFP
P70a-mGapt-deGFP
502065
$80
pTXTL-P70a-mGapt1
P70a-mGapt1
502064
$80
pTXTL-P70a-mRuby
P70a-mRuby
502066
$80
pTXTL-P70a-mmCherry
P70a-mmCherry
502063
$80
pTXTL-P70a-ntrC
P70a-ntrC
502071
$80
pTXTL-P70a-ompA-S24
P70a-ompA-S24
502070
$80
pTXTL-P70a-ompA-S38
P70a-ompA-S38
502078
$80
pTXTL-P70a-S32
P70a-S32
502075
$80
pTXTL-P70a-S32-ssrA
P70a-S32-ssrA
502076
$80
pTXTL-P70a-S38
P70a-S38
502077
$80
pTXTL-P70a-T3rnap
P70a-T3rnap
502081
$80
pTXTL-P70a-S54
P70a-S54
502079
$80
pTXTL-P70a-U3-deGFP
P70a-U3-deGFP
502083
$80
pTXTL-P70a-U4-deGFP
P70a-U4-deGFP
502084
$80
pTXTL-P70a-U5-deGFP
P70a-U5-deGFP
502085
$80
pTXTL-P70a-tagRFPT1
P70a-tagRFPT1
502080
$80
pTXTL-P70a-venus
P70a-venus
502086
$80
pTXTL-P70b-U3-deGFP
P70b-U3-deGFP
502090
$80
pTXTL-P70b-U4-deGFP
P70b-U4-deGFP
502091
$80
pTXTL-P70b-broccoli
pTXTL-P70b-broccoli
502122
$80
pTXTL-P70b-deGFP
P70b-deGFP
502087
$80
pTXTL-P70c-U4-deGFP
P70c-U4-deGFP
502096
$80
pTXTL-P70c-U3-deGFP
P70c-U3-deGFP
502095
$80
pTXTL-P70c-mGapt1
P70c-mGapt1
502093
$80
pTXTL-P70d-deGFP
P70d-deGFP
502097
$80
pTXTL-P70e-broccoli
pTXTL-P70e-broccoli
502123
$80
pTXTL-P70e-deGFP
pTXTL-P70e-deGFP
502124
$80
pTXTL-PLlacO1-deCFP
pTXTL-PLlacO1-deCFP
502126
$80
pTXTL-PLlacO1-deGFP
pTXTL-PLlacO1-deGFP
502125
$80
pTXTL-PLlacO1-tetR
pTXTL-PLlacO1-tetR
502127
$80
pTXTL-PLtetO1-deGFP
PLtetO1-deGFP
502098
$80
pTXTL-PLtetO1-deGFP-ssrA
PLtetO1-deGFP-ssrA
502099
$80
pTXTL-PLtetO1-deGFP-ybaQ
PLtetO1-deGFP-ybaQ
502100
$80
pTXTL-PLtetO1-lacO1
pTXTL-PLtetO1-lacO1
502128
$80
pTXTL-PLtetO1-mmCherry-ssrA
pTXTL-PLtetO1-mmCherry-ssrA
502133
$80
pTXTL-PLtetO1-tetR
pTXTL-PLtetO1-tetR
502129
$80
pTXTL-T3p-deGFP
T3p-deGFP
502104
$80
pTXTL-T7p11-deGFP
T7p11-deGFP
502109
$80
pTXTL-T7p11-mGapt
T7p11-mGapt
502110
$80
pTXTL-pC-mGapt
pC-mGapt
502102
$80
pTXTL-pCa-deGFP
pCa-deGFP
502103
$80
pTXTL-T7p14-aH
T7p14-aH
502113
$80
pTXTL-T7p14-aH-eGFP
T7p14-aH-eGFP
502114
$80
pTXTL-T7p14-deGFP
T7p14-deGFP
502111
$80
pTXTL-T7p14-deCFP
T7p14-deCFP
502140
$80
pTXTL-T7p14-mmCherry
T7p14-mmCherry
502141
$80
pTXTL-T7p14-luc
pTXTL-T7p14-luc
502130
$80
pTXTL-T7p16-deGFP
T7p16-deGFP
502115
$80
pTXTL-T7p14-mGapt
T7p14-mGapt
502112
$80
pTXTL-T7p16-mGapt
T7p16-mGapt
502116
$80
pTXTL-T7p7-deGFP
T7p7-deGFP
502105
$80
pTXTL-T7p7-mGapt
T7p7-mGapt
502106
$80
pTXTL-T7p8-deGFP
T7p8-deGFP
502107
$80
pTXTL-T7p8-mGapt
T7p8-mGapt
502108
$80
pTXTL-P70a-T7rnap
P70a-T7rnap
502082
$80
E. coli KL740 cI857+
E. coli KL740 cI857+ is mandatory for propagation of pTXTL-P70 vectors. The glycerol stock is delivered dry on a filter disc.
502000
$130
PLEASE NOTE: These products will no longer be available for purchase after August 5, 2024.

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