Nannochloropsis genetic elements

Promoters

Unidirectional

The lipid droplet surface protein (LDSP) promoter and elongation factor (EF) promoter are used for overexpression.

Bidirectional

Nannochloropsis oceanica possesses several bidirectional promoters that are utilized in genetic engineering, including VCP, ribi, and NR.

Resistance marker genes

Hygromycin (100-300 ug/ml) and zeocin (2-10 ug/ml) are the most widely used markers. Blasticidin has also been used in N. oceanica and N. gaditana, with concentrations between 25-100 ug/ml. Hygromycin and Blasticidin while effective have been reported to be leaky, either due to physiology or stability in media. G418 (250 ug/ml) and Nourseothricin (200 ug/ml) are very effective in N oceanica and diatoms. To increase stringency selection agent can be added to a transformation 12-24 hours in recovery.

Endogenous genes/auxotrophy

Nitrate assimilation genes (nitrate/nitrite reductase) are possible auxotrophic markers. Phytoene desaturase is another likely selectable gene.

Reporter genes

Fluorescent proteins

Several GFP variants have been used in Nannochloropsis including eGFP, cerulean, and venus; venus has the lowest background of the tested fluorescent proteins.

Luciferases

Firefly luciferase and NanoLuciferase have been codon optimized for N. oceanica and are used for reporter protein assays. Firefly luciferase is useful for extended timecourses. NanoLuciferase has very high signal useful for detecting protein expression. A cas9-Nanoluciferase was first utilized in N. oceanica, this fusion gene is detectable even with very low expression levels.

Chromoproteins

Expression of the purple chromoprotein (shPCP) is detectable by the naked eye.

Localization peptides

See Nannochloropsis_localization_peptides

Miscellaneous

2A peptides

Various 2A peptides have been tested in N. oceanica and N. salina. 2A peptides longer than the standard ~20 aa, of up to 60 aa are found to be even more effective. The most effective variants are P2A in N. oceanica, and E2A in N. salina.

Self-cleaving ribozymes

The hammerhead (HH) and hepatitis delta virus (HDV) ribozymes are likely active when expressed N. oceanica, as indicated by their use to produce functional sgRNAs when expressed from a Type II RNA polymerase promoter.

Codon optimization

The codon tables for N. gaditana and N. oceanica are available. These can be used with the online optimizer tool. Generated sequences should be checked for undesirable restriction sites.

Cloning systems

pNOC vectors

pNOC vectors originate at Michigan State University and are built using Nannochloropsis oceanica CCMP1779 genetic elements. It is built as a vector series with several reporter genes, resistance markers, and promoters (ie similar vectors containing variable genetic elements). There are compatible cut sites on both sides of reporter genes, AscI/HpaI on the 5' and MluI/NruI on the 3'.

Superstacking

The superstacking system enables the combination of two bidirectional promoter expression cassettes into a single vector, one cassette contains a resistance marker gene. The system utilizes a destination vector with bidirectional promoter expression cassette with resistance marker, and a gateway cloning site. An entry vector also contains a bidirectional promoter expression cassette. Reporter genes are present in both entry and destination vectors, and the destination vector series has a set of several resistance markers (HygR, BleR, BsdR, NeoR, NAT).

MoClo

Strategy for molecular assembly

Determine overhangs for each part type and assembly level.

Decide type IIS restriction Commonly used for MoClo Type IIS restriction enzymes: BsaI, BspQI/SapI, BsmBI, BbsI

Decide upon vector backbone.

Determine elements to generated in first trial.

Look at target sequences for problematic restriction sites.

Either synthesize or PCR target sequence.

This project will structured on the Free Genes and Bionode Projects and will thus be integrated with a decentralized DNA distribution system.

Elements for assembly

Backbone

Use of an open access backbone, either pUC19 or pOPEN.

Promoters

Bidirectional promoters an obstacle due both ends being connected to coding regions. Using the same overhang would lead to a loss in directionality and the coding regions being directly ligated together. Use of different overhangs would complicate standards. Use of second level assembly is a suggested solution. The level 1 product would contain a different type IIS restriction multicloning site next to the unused promoter end. Digestion would produce a promoter overhang and plasmid overhang, and a coding region and terminator could complete the level 2 assembly. Level 3 assembly requires a

Resistance markers

These may be good contributions to the Free Genes Project and should be designed to be useful across organisms.

References

Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., … Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074–2086. https://doi.org/10.1021/acssynbio.8b00251

Engler, C., Youles, M., Gruetzner, R., Ehnert, T.-M., Werner, S., Jones, J. D. G., … Marillonnet, S. (2014). A Golden Gate Modular Cloning Toolbox for Plants. ACS Synthetic Biology, 3(11), 839–843. https://doi.org/10.1021/sb4001504

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765