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Data page: Summary of our summer work

French Froggies : New Xenopus plasmids for creating multicellular systems

We developed and submitted to the registry 2 new plasmid backbones for creating multicellular synthetic systems and the corresponding biobricked promoters

• The pSC2+ with the CMV promoter: K812000
• The pSC2+ with pElastase promoter (pancreas specific): BBa_K812200
• And the Biobricked pElastase alones: BBa_K812233
• And with a fluorescent citrine to characterize the promoter pElastase in the frog:
• We also submitted a heat shock Xenopus promoter: BBa_K812331

We also prepared a series of fluorescent reporters with a kozak sequence for high expression in eukaryotes.

• A kozak-citrine in pSB1C3: K812030
• A kozak-sfGFP in pSB1C3: K812031
• A kozak-mCFP in pSB1C3: K812032

And provided them directely cloned in pSC2+ for direct expression in Xenopus or chicken:

• The Citrine in pSC2+: K812130
• The mCFP in pSC2+: K812132
• The sfGFP in pSC2+: K812133

All these plasmids can be directly injected in to the Xenopus embryo after a miniprep, and are designed for rapid testing of multicellular devices. They contain all sequences necessary for a tissue specific expression, as well as debugging tools. Several plasmids can be co-injected, allowing ratio adjustment and linking the system do modeling.Once the system is tested and debugged, the final system can be implemented by chromosomal insertion.

Characterization

The auxin system

Auxin production device

We adapted the auxin production module created by the Imperial College 2011 team for it to be used in eukaryotes. We made a system designed for coinjection, and a system which works with a single “operon” using pep2A. This is a short self-cleaving peptide, which will cut a nascent peptide chain to make 2 proteins, insuring stochiometry of the products in the cell. These auxin production devices are designed to communicate through the organism with the auxin reception device.

Fig 3: Schematic representation of the Auxin two-step production cassette.

• K812020: IAAH enzyme that catalyzes the second and last step towards auxin production
• K812021: IAAM enzyme that catalyses the transformation of Tryptophan to Inodle-3-acetamide
• K812014: Auxin production cassette containg both enzymes above which is monocystronic

Auxin reception device

The auxin reception device is composed of two components : Tir1, an auxin dependant ubiquitin ligase, and AID – a peptide that is recognized and ubiquitinated specifically by Tir1, when auxin is present. By fusing AID to any protein, this protein will be degraded along with the AID tag when auxin is added to the medium.

Fig 2: schemetic representation of the Auxin-receiver system

• K812010: GFP fusion with the ubuquitinase E3 OsTirI recoginition domain which a main part of the degron system
• K812012: OsTirI Ubiquitinase E3 for AID tagged protein degradation in the presence of auxin
• K812013: The Auxin reciever system golden gate-assembled containing GFP-AID OsTirI polysistronic system for auxin detection in tadpole

Goldenbricks

Summary

Fig 4: Summary of the GoldenBrick procedure

The GoldenBrick is a new assembly method for the partsregistry. If you want to know more, see this page.

Our favourites parts

• K812050: A GoldenBricked version of pSB1C3 with J04450 as negative cloning control
• K812051: A GoldenBricked version of pSB1K3 with J04450 as negative cloning control

The other parts we have created

• K812050: A GoldenBricked version of pSB1C3 with J04450 as negative cloning control
• K812051: A GoldenBricked version of pSB1K3 with J04450 as negative cloning control
• K812053: A GoldenBricked version of the strong RBS B0034
• K812054: A GoldenBricked version of the RFP E1010
• K812055: A GoldenBricked version of the terminator B0015
• K812056: A GoldenBricked version of the pLac R0010 promoter
• K812057: A GoldenBricked of an sfGFP protein
• K812058: A GoldenBricked of medium strenght RBS J61107
• K812059: A GoldenBricked of week RBS J61117