Parts
New parts submitted to the registry
| Parts name | Type | Part number |
|---|---|---|
| RBCS2i1 | Protein domain | BBa_K2703000 |
| pSAD | Regulatory | BBa_K2703002 |
| 3'LTR_Cr | Regulatory | BBa_K2703003 |
| 5'LTR | Regulatory | BBa_K2703004 |
| paromycin resistance gene | Coding | BBa_K2703008 |
pSAD
pSAD is a constitutive promoter in Chlamydomonas reinhardtii. It is is a high expression constitutive promoter that controls the expression of the gene encoding for a ferrodoxin-binding protein of photosystem I. This pSAD promoter was submitted in 2014 by the Concordia team (BBa_K1547005). To be compatible with the PhytoBrick standard (RFC1000), an illegal BpiI restriction site was removed (A289T). We cloned pSAD by PCR amplification in A2-A3 fusion site to use it for our retrotransposon design. The acceptor plasmid is the MoClo Universal Level 0 plasmid (pL0): pAGM9121. We sequenced again the sequence to be sure there are no unwanted mutations.
Role in our project
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be used as a constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution in the Chlamydomonas. It is part of our reporter system to characterize the activity and transposition rates.paromycin resistance gene
The sequence of the paromomycine-resistance marker is an aminoglycoside antibiotic. The aphVIII gene from Streptomyces rimosus encodes an aminoglycoside 3’-phosphotransferase gene (aph). From the aphVIII gene sequence of the BioBrick BBa_K1884013 (iGEM16_SZU-China) BpiI estriction sites were removed ( G551C) by PCR point mutation. We cloned aphVIII by PCR amplification in B4-B5 position of the MoClo standard2 to use it in our retrotransposon design. Acceptor plasmid is the MoClo Universal pL0 acceptor plasmid pAGM91212. We sequenced again the sequence to be sure there is no unwanted mutations.
Role in our project
Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intended to be used as a reporter gene in the CARGO to test the functionality of our synthetic retrotransposons for directed evolution in C. reinhardtii.RBCS2i1
This is the second intron of the rubisco protein from Chlamydomonas reinhardtii.
3'LTR_Cr
Long-Terminal-Repeat in the 3'end of a retrotransposon (or retrovirus). It contains the information necessary to start the reverse transcription.
5'LTR
Long-Terminal-repeat in the 5'end of a retrotransposon (or retrovirus). It contains the regulatory elements to start the transcription of the retrotransposon.
Parts used in our project but not submitted to the registry
| Parts name | Type | Part number |
|---|---|---|
| otsA | Coding | Bba_K2703001 |
| pL0-pSAD_A2-B1 | Regulatory | Bba_K2703005 |
| otsB | Coding | Bba_K2703006 |
| Bba_K2703006 | Regulatory | Bba_K2703007 |
| 5' UTR pSAD | Regulatory | Bba_K2703009 |
| Gag Pol | Coding | Bba_K2703010 |
| pAGM9121 | backbone | Bba_K2703011 |
