Parts overview
These pages contain each part that was utilized to develop AlgaGenix.
A wide range of Basic Parts and their subsequent Composite Parts have been designed and registered in order to create AlgaGenix. All of these components have been optimized for expression in the microalgae model organism Chlamydomonas reinhartii and conceived to be used with the MoClo technique, based on the Golden Gate assembly using type II S restriction enzymes.
All sequences were synthetised by the sponsor Twist Bioscience as well as backbone vectors offered by the Computational and Synthetic Biology research group of Center for Rresearch in Agricultural Genomics (CRAG)
Additionally, we have also added to the registry the different parts from the MoClo kit we have used. We believe it could assist future iGEM teams in understanding the benefits of using this technique alongside the wide range of pre-designed components to work with.
We have created a Part Collection containing the newly synthesized Nitrates Sensor (number) followed by the designed genetic constructs (numbers) that will enhance nitrates absorption and its consequent transformation into cytokines.
You can find below the different parts created by AlgaGenix. We anticipate and hope that this Part Collections will be useful and help future iGEM teams in engineering Chlamydomonas reinhardtii and exploiting MoClo's adaptability to tackle any synthetic biology problem. If you need more details and precisions about our parts, please do not hesitate contacting us at algagenix@gmail.com
| Part | Registry code | Registry code | Part name | Description |
|---|---|---|---|---|
| basic | Promoter | BBa_K4770007 | PPSAD | Constitutive promoter, active under all circumstances and independent to stimulus. |
| basic | CDS | BBa_K4770002 | NR, Nitrate Reductase | Coding sequence. Enzyme responsible for the first reduction step from nitrate to nitrite. |
| basic | CDS | BBa_K4770003 | NiR, Nitrite Reductase | Coding sequence. Enzyme responsible for second reduction step from nitrite to ammonium in the chloroplast. |
| basic | CDS | BBa_K4770004 | GS, Glutamine Synthetase | Coding sequence. Enzyme responsible for the ammonium incorporation into carbon skeletons. This enzyme has two forms: cytosolic and one localized in the chloroplast. This basic part is designed to be located in the cytoplasm. |
| basic | Transit Peptide | BBa_K4770010 | Cp | Coding sequence. Transit peptide, marks proteins to be delivered to the chloroplast surface by cytosolic chaperones. |
| basic | CDS | BBa_K4770005 | IPT, Adenylate isopentenyl transferase | Coding sequence |
| basic | CDS | BBa_K4770006 | LOG, cytokinin phosphoribohydrolase ‘Lonely guy’ | Coding sequence |
| basic | Linker | BBa_K4770012 | F2A | Self-cleaving peptide, used to link CDS and reporter |
| basic | Reporter | BBa_K4770011 | NanoLuc luciferase | Luciferase enzyme able to produce high intensity, glow-type luminescence. |
| basic | Terminator | BBa_K4770008 | TPSAD | Constitutive terminator. Causes transcription to stop. |
| basic | Resistance | BBa_K4770009 | HygroR Gene | Hygromycin resistance. Gene that confers ability to inactivate hygtomycine through phosphorylation. Can be used for colony’s selection. |
| basic | Fluorescent Tag | BBa_K4770013 | mCherry Gene | Red Fluorescent Protein (RFP) used as a fluorescent reporter tag. |
| basic | Fluorescent Tag | BBa_K4770014 | mVenus Gene | Hygromycin resistance. Gene that confers ability to inactivate hygtomycine through phosphorylation. Can be used for colony’s selection. |
| basic | Promoter | BBa_K4770024 | pNIT | Nitrate-dependant promoter, highly induced by nitrated and repressed by ammonia. |
| composite | Coding | BBa_K4770015 | Cp-GS | Coding sequence. Enzyme responsible for the ammonium incorporation into carbon skeletons. This enzyme has two forms: cytosolic and one localized in the chloroplast. This basic part is designed to be located in the chloroplast (Cp number + GS number). |
| composite | MoClo’s Level 1 | BBa_K4770018 | NR Level 1 | Level 1 plasmid including Ppsad promoter, NR cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of NR, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770019 | NiR Level 1 | Level 1 plasmid including Ppsad promoter, NiR cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of NiR, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770020 | GS Level 1 | Level 1 plasmid including Ppsad promoter, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770021 | Cp-GS Level 1 | Level 1 plasmid including Ppsad promoter, chloroplast transit signal, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770022 | IPT Level 1 | Level 1 plasmid including Ppsad promoter, NR cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of NR, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770023 | LOG Level 1 | Level 1 plasmid including Ppsad promoter, NR cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of NR, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s individual gene Level M | BBa_K4770028 | GS Level M | Assembly of different transcriptional units into the same vector: HygroR cassette, pCM1:GS, mVenus cassette, and mCherry cassette. |
| composite | MoClo’s individual gene Level M | BBa_K4770029 | Cp-GS Level M | Assembly of different transcriptional units into the same vector: HygroR cassette, pCM1:CpGS, mVenus cassette, and mCherry cassette. |
| composite | MoClo’s Level 1 | BBa_K4770021 | Cp-GS Level 1 | Level 1 plasmid including Ppsad promoter, chloroplast transit signal, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s individual gene Level M | BBa_K4770030 | IPT Level M | Assembly of different transcriptional units into the same vector: HygroR cassette, pCM1:IPT, mVenus cassette, and mCherry cassette. |
| composite | MoClo’s individual gene Level M | BBa_K4770031 | LOG Level M | Assembly of different transcriptional units into the same vector: HygroR cassette, pCM1:LOG, mVenus cassette, and mCherry cassette. |
| composite | MoClo’s gene pairs Level M | BBa_K4770032 | NR-NiR Level M | Assembly of different transcriptional units into the same vector. Designed “Gene-pairs Level Ms”, containing HygroR cassette, level 1 for two different genes (pCM1:NR + pCM1 NiR in this case) mVenus cassette, and mCherry cassette. With this vector, we aim to see the effects of overexpressing the first two genes of the nitrates assimilation pathway constitutively and simultaneously. |
| composite | MoClo’s gene pairs Level M | BBa_K4770033 | GS-Cp-GS Level M | Assembly of different transcriptional units into the same vector. Designed “Gene-pairs Level Ms” containing the HygroR cassette, level 1 for two different genes (pCM1 GS+ pCM1 Cp-GS), mVenus cassette, and mCherry cassette. With this vector, we aim to see the effects of simultaneously overexpressing both dorms (cytosoloic and in the chloroplast) of the Glutamamte Synthetase, enzyme responsible for last step of nitrates assimilation. |
| composite | MoClo’s gene pairs Level M | BBa_K4770034 | IPT-LOG Level M | Assembly of different transcriptional units into the same vector. Designed “Gene-pairs Level Ms”containing the HygroR cassette, level 1 for two different genes (pCM1 IPT + pCM1 LOG in this case), mVenus cassette, and mCherry cassette. With this vector, we aim to see the effects of overexpressing two of the most important enzymes on the cytokine synthesis pathway. |
| composite | Resistance cassette | BBa_K4770016 | HygroR cassette | Constitutive promoter paired to hygromycin resistance gene to enhance and maintain its expression activated. Resistance cassettes can be used as a selectable marker for colonies and ensure correct identification of colonies that have successfully integrated the vectors into their genomes. |
| composite | MoClo’s Level 1 | BBa_K4770021 | Cp-GS Level 1 | Level 1 plasmid including Ppsad promoter, chloroplast transit signal, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | Constitutive reporter | BBa_K4770017 | mCherry cassette | Constitutive promoter paired to mCherry gene to enhance and maintain its expression activated. Used to measure expression levels depending on the site of insertion inside the genome. |
| composite | MoClo’s Level 1 | BBa_K4770021 | Cp-GS Level 1 | Level 1 plasmid including Ppsad promoter, chloroplast transit signal, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | Inducible reporter | BBa_K4770021 | Cp-GS Level 1 | Level 1 plasmid including Ppsad promoter, chloroplast transit signal, GS cds linked through F2A to NanoLuc and Tpsad terminator. This Composite Part allows constitutive expression of GS in the cytoplasm, which can be confirmed through NanoLuciferase signal. |
| composite | MoClo’s Level 1 | BBa_K4770025 | mVenus cassette | Nitrates-dependent promoter paired to mCherry gene. Used to measure expression levels depending on nitrates concentration in the media. Strongly activated by nitrates and inhibited by ammonium. |