The Nucleus v0.1.0 distribution plate is the first release of the physical DNA encoding Nucleus content components and modules, available under the OpenMTA.

Table of Contents

• Plate Map
• Contents
• Technical Information
• Getting Started
• Notes
• Errata
• Other Resources

Key Information

Backbone	pOpen v3
Antibiotic Resistance	Amp
Concentration (fmol)	10
Total volume (ul)	10
Sequence Information	[Github]

Plate Map

Contents

The content of the plate is described in the DNA Distribution document. In general, the plate is arrayed by plasmid category:

1. PURE genes occupy the first three rows (A-C), in the gene ordering originally published by Shimizu, et al (2001).
2. Row D contains alternative versions of the PURE genes, such as different designs for the two-subunit tRNA synthetases, and metabolic enzymes sourced from different organisms as have been used in the literature.
3. Row E contains open measurement reporters.
4. Row F contains T7 promoter variants engineered to span two orders of magnitude of expression, for direct testing or use as a MoClo level 0 promoter part.
5. Row G contains inducible modules for use in the PURE cell free system.
6. Row H contains several basic MoClo level 0 parts for assembly of level 1 transcription units.

Technical Information

• All DNA is encoded on the high-copy pOpen v3 plasmid, in the Nucleus DNA architecture.
• All plasmids on the plate are Ampicillin / Carbenicillin resistant.
• Each construct is normalized to 10 fmol / uL and provided as 10 uL total.
• Plates are sealed with slit silicon plate seals and lids. DNA can be extracted through the slits in the seal without removing it.
• Multichannel pipetting through the slit seal is very difficult.
• Exercise caution if removing the seal—be very gentle, or the vacuum produced will pull sample from the well and potentially cross-contaminate the wells.
• DNA may have shifted in transit—spin down the plate before using it in downstream work.

Getting Started

We recommend sub-cloning the DNA into an appropriate cloning strain such as DH5-alpha or NEB Stable, and preparing more high concentration DNA for downstream use.

1. Spin down the plate in a tabletop plate spinner, or a centrifuge at 2000g for 1 minute.
2. Dilute the DNA by adding 90 ul of TE buffer or nuclease-free water to each well, through the slit.
1. Pipette mix while adding the additional solution to each well.
3. Briefly spin as before.
4. Transform 2 ul of each desired construct into DH5-alpha or NEB Stable competent cells, following the manufacturer’s protocol.
5. Prepare DNA using an appropriate kit or protocol.
6. Store original DNA at -20ºC.

Notes

1. DNA may be resuspended and transformed by multichannel pipette, by slowly and carefully removing the silicon seal after spinning down.
2. DNA may be kept at a higher concentration for direct use in PCR or Golden Gate reactions, although the reaction volume will be limited.
3. TE buffer is our preference for preserving DNA quality over extended periods of time, and will not interfere with downstream transformation or assembly reactions.

Errata

• Components highlighted in red are either not included on this plate distribution, or have known problems.
• ArgRS, LeuRS, and T7RNAP are being resynthesized. Variants of these genes are available separately under the OpenMTA, but not in the Nucleus DNA architecture.
• plamGFP-TXTL and pOpenv3-MCL0 are not yet available as part of the collection.
• The lacI lac-repressor protein was mis-synthesized; the construct in this well encodes a second copy of tetR.

Other Resources

• Fluorescence parameters for the open reporters in the distribution are available at FPBase: https://www.fpbase.org/collection/2724/.