Contributions

We put together several basic parts using the Chlamydomonas reinhardtii MoClo toolkit and plant MoClo toolkit.

Nitrogen Parts

We created the following basic parts for our nitrogen pathway:

Ammonium monooxygenase (AMO)
Hydroxylamine oxidoreductase (HAO)
Nitrous Oxide Reductase (nosZ)

Original Gene	Accession Number	Description	Part Number	Link to Part Page
Ammonia monooxygenase (AMO)	EIC29219.1	Takes ammonia and converts it to NH2OH	BBa_K4707000	parts.igem.org/Part:BBa_K4707000
Hydroxylamine oxidoreductase (HAO)	CAI8898772.1	Takes NH2OH and converts it to N2O	BBa_K4707001	parts.igem.org/Part:BBa_K4707001
Nitrous Oxide Reductase (nosZ)	WP_013993592.1	Converts the N2O to N2	BBa_K4707002	parts.igem.org/Part:BBa_K4707002

Nitrogen Devices in L1 Plasmids

AMO, HAO, and Nos Z — This table image describes our L1 plasmid builds. Our genes of interest are in the B3-4 sections. The His, Flag, and Clover tags identify successful transformations. The Clover tag gives off a green fluorescent light. Our Psad promoter and Tsad terminator are at the beginning and end of each part.

Antibiotic Resistance Devices in L1 Plasmids

Antibiotic Resistance Crnptii and aadA — The two constructs in the image above are for antibiotic resistance, which allows us to select the successfully transformed algae. Crnptii provides kanamycin resistance, and aadA provides spectinomycin resistance. Green algae that successfully take up our nitrogen and phosphorus genes, which include one of these antibiotic resistance constructs, are able to survive the antibiotic that the construct codes resistance to. Green algae that successfuly took up the genes can then be identified by seeing whether they survived the antibiotic or not.

Ammonium Monooxygenase (AMO)

Our ammonium monooxygenase gene is from the organism Methylomicrobium album BG8. It codes for the enzyme that converts nitrate to NH2OH. This gene works in combination with our other genes that code to take bioavailable nitrate to dinitrogen gas that will be released into the atmosphere. The NH2OH is an intermediate in our process. This sequence is meant to be expressed in the green algae Chlamydomonas reinhardtii.

pL0 AMO Map from SnapGene — This is a SnapGene plasmid map of our L1 AMO part.

Hydroxylamine Oxidoreductase (HAO)

Our Hydroxylamine Oxidoreductase gene is from the organism Methylocaldum szegediense. HAO is an enzyme that works to change NH2OH to nitrous oxide. This allows for a nitrifier denitrification pathway to continue in another organism as we insert it. It works together with AMO and nitrous oxide reductase to take nitrate from ammonia to dinitrogen gas. This sequence is meant to be expressed in the green algae Chlamydomonas reinhardtii.

pL0 HAO Map from SnapGene — This is a SnapGene plasmid map of our L1 HAO part.

Nitrous Oxide Reductase (nosZ)

Our Nitrous Oxide Reductase gene came from the organism Zobellia galactanivorans. It changes nitrous oxide to dinitrogen gas. This allows for a nitrifier denitrification pathway to continue in another organism as we insert it. It works together with AMO and HAO to take nitrate to ammonia to dinitrogen gas. This sequence is meant to be expressed in the green algae Chlamydomonas reinhardtii.

pL0 Nos Z Map from SnapGene — This is a SnapGene plasmid map of our L1 nosZ part.

L2 Nitrogen Plasmid Map from SnapGene — This is a SnapGene plasmid map of our L2 Nitrogen, which contains our L1 AMO, HAO, and nosZ parts.

Fluorescent L2 Nitrogen transformed microalgae under a microscope — This image shows *Chlamydomonas reinhardtii* colonies that successfully took up our L2 Nitrogen plasmid DNA, a combination of our L1 AMO, HAO, and nosZ parts, which contained a part for fluorescence. Colonies with fluorescence indicate a successful uptake, while non-fluorescent colonies indicate unsuccessful uptake of the DNA plasmid.

Phosphorus Parts

This image shows our L1 Psr design. The primer extends to B3. The Level 0 Psr plasmid is from the Baker Lab, described in Slocombe et al. 2023, Front Plant Sci doi.org/10.3389/fpls.2023.1208168

Plant MoClo Toolkit L1 Plasmids

Plasmid	Description
pICH47732	Level 1 position 1F acceptor plasmid
pICH47742	Level 1 position 2F acceptor plasmid
pICH47751	Level 1 position 3F acceptor plasmid
pICH47761	Level 1 position 4F acceptor plasmid
pICH47772	Level 1 position 5F acceptor plasmid
pICH86966	Level 2 with kanR driven by nos promoter
pICH54022	“Dummy” part level 1 position 2

Toolkit: Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 2014 Nov 21;3(11):839-43. doi: 10.1021/sb4001504. Epub 2014 Feb 20. PMID: 24933124.

The table above shows the L1 plasmids that we used from the Plant MoClo Toolkit. These plasmids were used as vectors for the devices. We were able to build the promoter, indicator tags, and terminators in these L1 plasmids. We used the BsaI Golden Gate enzyme to insert our building blocks into the L1 plasmids.

Phosphorus Paper: Slocombe SP, Zúñiga-Burgos T, Chu L, Mehrshahi P, Davey MP, Smith AG, Camargo-Valero MA and Baker A (2023) Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake. Front. Plant Sci. 14:1208168. doi: 10.3389/fpls.2023.1208168

Chlamy MoClo Parts

Plasmid with Part	Part Location	Part Name
pCM0-10	A1-B1	PPSAD (Pro + 5'UTR)
pCM0-16	A1-B2	PPSAD (Pro + 5'UTR)
pCM0-41	B2	βTUB2 Intron 1 (intron as 5'UTR)
pCM0-45	B2	Clover (i2)-Strep (N-ter, CDS)
pCM0-59	B3	2A B3
pCM0-69	B3-B5	amiRNA (SpeI internal site, CDS)
pCM0-75	B3-B5	CrnptII (KanR, CDS)
pCM0-76	B3-B5	aadA (SpecR, CDS)
pCM0-87	B4	2A B4
pCM0-94	B5	3Flag (with internal Lys, C-ter, CDS)
pCM0-95	B5	6His (C-ter, CDS)
pCM0-97	B5	3*Myc-tag (C-ter, CDS)
pCM0-98	B5	HA (C-ter, CDS)
pCM0-105	B5	Clover (i2)-Strep (C-ter, CDS)
pCM0-114	B6-C1	TSAD (3'UTR + Ter)

Toolkit: Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii Pierre Crozet, Francisco J. Navarro, Felix Willmund, Payam Mehrshahi, Kamil Bakowski, Kyle J. Lauersen, Maria-Esther Pérez-Pérez, Pascaline Auroy, Aleix Gorchs Rovira, Susana Sauret-Gueto, Justus Niemeyer, Benjamin Spaniol, Jasmine Theis, Raphael Trösch, Lisa-Desiree Westrich, Konstantinos Vavitsas, Thomas Baier, Wolfgang Hübner, Felix de Carpentier, Mathieu Cassarini, Antoine Danon, Julien Henri, Christophe H. Marchand, Marcello de Mia, Kevin Sarkissian, David C. Baulcombe, Gilles Peltier, José-Luis Crespo, Olaf Kruse, Poul-Erik Jensen, Michael Schroda, Alison G. Smith, and Stéphane D. Lemaire ACS Synthetic Biology 2018 7 (9), 2074-2086 DOI: 10.1021/acssynbio.8b00251

The above table shows the parts that we used from the Chlamydomonas MoClo (Modular Cloning) Toolkit, which provides various building blocks to build a device. This kit was used to take our genes of interest and insert them into a module that eventually became a device. We included a promoter, indicators, and terminators from the toolkit. This means that each plasmid only contains one piece of the device that we are building.

Become a Sponsor