iGEM 2021: Mingdao

Mingdao

MEASUREMENT

CHALLENGES IN MEASUREMNETS

Characterizing parts and calibrating data in measurements are encouraged in iGEM projects. However, the challenges will be met in cell-based system, just like we did in our project, including (1) variable promoter activities in different cell culture environments and conditions in reporter assays, (2) lack of suitable plasmid vectors or antibiotic selection, (3) proteins in the cells are unstable, toxic, insoluble or misfolding, (4) virus production and purification, (5) biosafety level concerns, etc.

Cell-free in vitro transcription-translation (TXTL) system has been developed and increasingly used in research studies and iGEM projects such as Imperial in 2007, Caltech in 2014, UCC_Ireland and Lethbridge and EPFL in 2017, Stanford and ETH_Zurich, EPFL and Wageningen_UR in 2019, Waseda in 2020, in which either using an expensive commercial kit (e.g., PURExpress®) or describing incomplete or complicated methods. Therefore, we are encouraged and determined to develop an easy-to-use methodology in TXTL. And we’ve applied this methodology in lots of our projects including our measurements on our parts and functional assays.

IN VITRO TRANSCRIPTION-TRANSLATION (TXTL)

In vitro transcription and translation (TXTL) is a convenient cell-free system that has increasingly been developed to apply in synthetic biology^1,2. In addition to achieve biosafety level, TXTL becomes powerful in prototype characterization of genetic parts, devices and circuits. Moreover, TXTL is particularly useful to express and purify proteins which are toxic, insoluble or unstable in cell-based system. Furthermore and amazingly, Dr. Vincent Noireau’s lab has demonstrated cell-free TXTL application in infectious bacteriophage production, in which T7 phage (40kbp, 77 genes, dsDNA) and T4 phage (170kbp, 289 genes, dsDNA) genome replication, synthesis, assembly can be performed in vitro just in a single test tube^3,4.

CELL-FREE TXTL as EASY as 1,2,3

↓Prepare your materials

↓Following the protocol

↓And, just do your assays

We’ve used TXTL in part characterizations (BBa_K3376000, BBa_K3728001, BBa_K3728003, BBa_K3728005, BBa_K3728006, BBa_K3728008), protein expression and purification, functional assays and bacteriophage synthesis.

Material Preparation

Based on the Mark Rustad’s T4 phage paper⁴ and more detailed description in Zachary Z Sun’s work⁵, we did a slight modification and a simple version as follows.

DNA

The PCR-amplified DNA fragments or the mini-prep of plasmid DNAs (50- 200 ng/μl) or gDNAs (300-500 ng/μl) can be applied in TXTL cell-free system.

Bacterial extracts

Prepared as follows:

↓Take the bacteria of E. coli Rosetta 2(DE3) or Salmonella Typhi (for example) as 1:200 dilution into 50mL LB, shaking at 200 rpm, 37°C for 3- 4hr until OD600 between 1.5 and 2.0.

↓Centrifuge to get the pellet at 5,000xg, 4°C for 12min, followed by wash with 3 times with 20ml of S30A Buffer (14mM Mg-glutamate, 60mM K- glutamate, 50mM Tris at pH= 7.7, 2mM DTT).

↓Resuspend in 1ml of S30A Buffer and vortex until the mixture is homogeneous. (Keep the tube on ice as quickly as possible)

↓Add 5 volumes of 0.1-mm Glass Beads (Scientific Industries, Inc.) for cell disruption. Vortex 10 times at full speed for 30sec with an interval of 30sec on ice.

↓After centrifuging at 12,000xg, 4°C for 10min, transfer the supernatant to a new eppendorf.

↓Measure protein concentration and make sure the bacteria extracts is over 10 mg/ml.

↓Store at -80°C until use.

Amino acid solution;

Make a solution at 15mM for each of 20 canonical amino acids.

Energy buffer

The 10X Energy Buffer is composed of 500mM HEPES at pH=8, 15mM ATP, 15mM GTP, 9mM CTP, 9mM UTP, 2mg/ml tRNA solution, 2.6mM coenzyme A, 3.3mM NAD, 7.5mM cAMP, 0.68mM folinic acid, 10mM spermidine, and 300mM 3-PGA.

Chemicals or cofactors

Make stocks of 5M K-glutamate, 250mM Mg-glutamate, 750mM Maltose, 40% PEG 8000.

Protocol

↓Mix the materials as shown in Table 1.

↓Incubate at 30°C (or an indicated temperature) overnight or for an indicated time.

↓Measure protein activity or perform functional assay depending on your experiment.

*The reaction volume can be adjusted or aliquoted for reaction in a smaller volume. *If any problems in use, don’t hesitate to contact us: biolab@ms.mingdao.edu.tw

IN VITRO REPORTER ASSAY

To test the GFP (BBa_K3728005) and RFP intensities (BBa_K3728003 and BBa_K3728006) driven by ldhp or lacp, the indicated plasmids were put into TXTL reaction and measured the fluorescence (Fig. 1). The strong GFP fluorescence can even be visualized by naked eyes under a Blue LED Illuminator. Compared the activities of ldhp to lac promoter (lacp), lacp is inhibited in TXTL because the extracts of E. coli Rosetta 2 (DE3) contains LacI repressor, which can be relieved by IPTG induction or using E. coli DH5α as extracts.

Figure 1 | Promoter activities on pTol2 vector in TXTL. GFP fluorescence was measured at Ex/Em = 488/530 using a microplate reader of BioTek Synergy H1. RFP was at Ex/Em = 586/611. KanR/pTol2 in TXTX was set as a background control. AU means arbitrary unit. (a) ldhp-GFP-Tr/pTol2 activity in TXTL. The inset photo was captured under a blue LED light. (b) ldhp-RFP-Tr/pTol2 and J04450/pTol2 (i.e., lacp-RFP-Tr) in TXTL.

IN VITRO PROTEIN EXPRESSION & PURIFICATION

Tol2 Transposase

T7-driven Tol2 transposase (TPase) gene (BBa_K3728001) was expressed in TXTL reaction with the bacterial cytoplasmic extracts prepared from IPTG-induced E. coli Rosetta 2(DE3) cells. The His-tagged proteins were further purified through Nickel column. The protein concentration was measured and analyzed on SDS-PAGE and Coomassie Blue Staining (Fig. 2). The protein was shown at around 70 kDa as the same size as the predicted TPase protein (664 a.a., 75 kDa). The Elution #4, #5 and #6 were collected and used for further studies.

Figure 2 | His-Tol2 transposase was expressed in TXTL and purified by Nickel column. 10 μg of protein lysates were analyzed by SDS-PAGE and Coomassie Blue Staining using 4–12% gradient gel (NuPAGE™, Thermo Fisher Scientific Inc.) Lane: (1) PageRuler™ Prestained Protein Ladder, (2) E. coli Rosetta 2(DE3) cell extracts (no DNA control), (3) total lysates in TXTL, (4) flow-through, (5) wash-through, (6) Elution #4, (7) Elution #5, (8) Elution #6, (9) Elution #7, (10) Elution #8, (11) Elution #9.

Phi29 DNA polymerase and RCA assays

To characterize the function of recombinant Phi29 DNA polymerase (BBa_K3728008), TXTL cell-free system can solve the problem caused by the difficulty in bacterial transformation or without suitable plasmid vectors. We took the DNA of ldhp-Phi29 DNA pol-Tr/pTol2 mixed into TXTL reaction with Salmonella extracts. The recombinant His-tagged Phi29 DNA polymerase protein was purified through Nickel column and analyzed by SDS-PAGE and Coomassie Blue Staining (Fig. 3). The isolated proteins in Elution #4 and #5 were at the size of around 70 kDa as predicted (His-Phi29 DNA polymerase: 590 amino acids, 68 kDa) and collected for the following studies.

Figure 3 | His-Phi29 DNA polymerase was expressed in TXTL using Salmonella extracts and purified by Nickel column. 5 μg of protein lysates were analyzed by SDS-PAGE and Coomassie Blue Staining using 4–12% gradient gel (NuPAGE™, Thermo Fisher Scientific Inc.) Lane: (1) PageRuler™ Prestained Protein Ladder, (2) Salmonella cell extracts (no DNA control), (3) total lysates in TXTL, (4) flow-through, (5) wash-through, (6) Elution #3, (7) Elution #4, (8) Elution #5, (9) Elution #6, (10) Elution #7, (11) Elution #8.

We performed RCA by mixing a circular ssDNA and primer in the buffer with our purified Phi29 DNA polymerase (Φ29) from TXTL by Salmonella extracts or a commercial recombinant Φ29 from New England Biolabs Inc. (NEB) as a positive control. The Φ29 enzymes were diluted with various factors in the assay. After incubation at 30°C for 2 hours, the RCA products were stained with EvaGreen DNA dye and subjected to a microplate reader to measure signals at Ex/Em=488/530 nm. And the fold changes in fluorescence intensity were calculated by dividing the values from Φ29-untreated groups (as controls). As shown in Fig. 4, a 12-fold change was achieved with our Φ29, indicating the functionality and the activity of ldhp-Phi29 DNA pol-Tr/pTol2 that are comparable to the commercial NEB Φ29 enzymes. Moreover, the green fluorescence can readily be seen with a blue led light, even using a 4 times diluted Φ29, proving the super high processivity of Phi29 DNA polymerase in DNA amplification.

Figure 4 |RCA assay using TXTL-expressed (MINGDAO) or commercial (NEB) Phi29 DNA polymerase (Φ29). The commercial Φ29 was purchased from NEB with a defined activity by units. The control was set without Φ29 treatment. The concentration and dilutions of enzymes were, respectively, 4, 2, 1 μg/μl for MINGDAO Φ29 and 1, 0.5, 0.25 units for NEB Φ29. The EvaGreen DNA binding signals were read at Ex/Em=488/530 nm in BioTek Synergy H1 Microplate Reader.

IN VITRO BACTERIOPHAGE SYNTHESIS

We extracted the genomic DNAs of two of our isolated Salmonella phages and confirmed the gDNA integrity by running electrophoresis on 0.7% agarose gel (Fig. 5a). No plaque was observed using the extracted DNAs in the plaque assay (Fig. 5b), indicating no live phage contamination is in isolated gDNA extracts. Further, the extracted phage gDNA is able to generate infectious particles in Salmonella plaque assay after subjected to TXTL (Fig. 5c), demonstrating phage DNA can be expressed, assembled and packaged in vitro. Taken together, we can perform phage gDNA extraction and genome packaging in TXTL in our lab.

Figure 5 |Salmonella gDNA extraction and packaging in TXTL. (a) The integrity of DNA isolated from two Salmonella enterica serovar Typhimurium phages (named ST1 and ST2) was checked by electrophoresis on 0.7% agarose gel. (b,c) Extracted DNA as a control or DNA in TXTL reaction was spread on the lawn of Salmonella culture in plaque assay.

REFERENCE

1. Marshall R, Noireaux V. Synthetic Biology with an All E. coli TXTL System: Quantitative Characterization of Regulatory Elements and Gene Circuits. Methods Mol Biol. 2018;1772:61-93. doi: 10.1007/978-1-4939-7795-6_4.

2. Tinafar A, Jaenes K, Pardee K. Synthetic Biology Goes Cell-Free. BMC Biol. 2019 Aug 8;17(1):64. doi: 10.1186/s12915-019-0685-x.

3. Shin J, Jardine P, Noireaux V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol. 2012 Sep 21;1(9):408-13. doi: 10.1021/sb300049p.

4. Rustad M, Eastlund A, Jardine P, Noireaux V. Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synth Biol (Oxf). 2018 Jan 22;3(1):ysy002. doi: 10.1093/synbio/ysy002.

5. Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. J Vis Exp. 2013 Sep 16;(79):e50762. doi: 10.3791/50762.

Team:Mingdao/Measurement