Resources

PLASMIDS

We receive very large numbers of requests for plasmid materials every month. Fulfilling the volume of requests is beyond our capacity. However, we’re very keen to see our research help your research. To enable this, we deposit all our plasmids at Addgene. You can find all of our published plasmids at https://www.addgene.org/Nicola_Patron/. Addgene stores, sequences and distributes plasmids on behalf of thousands of labs, helping scientists make research materials available to the community on a non-profit, cost-recovery basis (requestors pay a fee to cover shipping, handling, maintenance and quality assurance). Addgene has a streamlined Material Transfer Agreement (MTA) process and stays up-to-date with the ever-changing requirements of how to ship biomaterials to most countries. If you deposit your own plasmids, you will accrue points that allow you to request plasmids free of charge.

DNA Assembly

We use a Type IIS parallel DNA assembly method (also known as Golden Gate Cloning). We led the establishment of the PhytoBrick TypeIIS standard. We have also published a toolkit of basic parts for plants, described in Engleret al (2014) ACS Synthetic Biology doi: 10.1021/sb4001504.

For a detailed laboratory protocol for TypeIIS-mediated DNA-assembly, please see Cai et al (2020) Phytobricks: Manual and Automated Assembly of Constructs for Engineering Plants. Methods Mol Biol. 2020;2205:179-199. doi: 10.1007/978-1-0716-0908-8_11 . You can also download a help sheet on how to make new parts.

Genome editing

We have published a detailed protocol for designing and testing constructs for targeted mutagenesis in plants: Dudley et al (2022)  Cas9-Mediated Targeted Mutagenesis in Plants. Methods Mol Biol;2379:1-26. doi: 10.1007/978-1-0716-1791-5_1 . This manuscript includes detailed methods for transient agroinfiltration of Nicotiana leaves and protocols for preparing and transfecting mesophyll protoplasts.

Cell-free protein synthesis

For E. coli CFPS reactions we use a lab-made extract generated from BL21(DE3) E. coli cells based on a modified PANOx-SP formula (PEP, amino acids, NAD+, oxalic acid, spermidine, putrescine) as described in Dudley et al  (2020) Metab. Eng.,61,251–260.

For plant CFPS reactions, we use wheat germ cell-free expressions used the TNT® SP6 High-Yield Wheat Germ Protein Expression System L3260/L3261 (Promega) according to manufacturers’ instructions.

We published a high throughput approach, described in Dudley et al (2021)  Biofoundry-assisted expression and characterisation of plant proteins. Synth Biol. 6(1):ysab029. doi: 10.1093/synbio/ysab029

Synthetic Promoters

We have published a number of synthetic promoters for plants. These include minimal synthetic promoters that are activated by endogenous plant transcription factors described in:

Cai et al (2020) Rational design of minimal synthetic promoters for plants. Nucleic Acids Res. 2020 Dec 2;48(21):11845-11856. doi: 10.1093/nar/gkaa682 

Cai et al (2023) Tuning Plant Promoters Using a Simple Split Luciferase Method to Assess Transcription Factor-DNA Interactions. ACS Synth Biol. 2023 Nov 17;12(11):3482-3486. doi: 10.1021/acssynbio.3c00094 

We have also characterised a range of synthetic promoters that are activated by synthetic inducible and/or programmable transcription factors:

Kallam et al (2023) Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana. Plant Biotechnol J. 2023 Jul;21(7):1440-1453. doi: 10.1111/pbi.14048 PMID: 37032497

CODE

“## CRISPRanto“; a (Snakemake) pipeline for CRISPR offtarget detection used in Raitskin et al (2019)