Part:BBa_K1072010

Odr-10(diacetyl)GPCR with optimized codon

Odr-10(diacetyl)GPCR encodes a diacetyl-sensing G-protein coupled receptor, originating from the C. elegans.

If BBa_K1072010 was transformed into the yeast, the pheromone receptors will be actived and coupled to a heterotrimeric G protein. pathway is composed of Gpa1 (Gα), Ste4 (Gβ) and Ste18 (Gγ) subunits.The Gβγ particle transmits the signal to a MAP kinase module and finally follow a inducible promotor FUS1, which will links a reproter, such as GFP, His3 or LacZ.

Figure 1. a diagram of Odr-10, 7-transmembrane receptors.

We fuse BBa_K1072008 with plasma membrane protein,Odr-10 and expressed in yeast successfully figure 2.

Figure 2. fluorescence was detected around plasma membrane

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Team INSA-UPS France 2017 usage of BBa_K1072010 in Pichia pastoris strain : diacetyl detection by Odr-10 receptor by monitored by the activation of pFUS1 promoter

We wanted to build a gene with a diacetyl inducible expression using Odr-10/pFUS1 system.

Indeed, when diacetyl binds to Odr-10 (BBa_K431009) an activation cascade of Ste proteins (endogenous to P. pastoris) will lead to the binding of Ste12 on the pFUS1 promoter, and so to the transcription of pFUS1 reporter gene (RFP, BBa_J04450).

To characterize the detection of diacetyl by Odr-10, we designed the following construction (see Figure 1):

Figure 1: Construction to characterize pFUS1 promoter A diacetyl receptor (Odr-10) is expressed under the control of a constitutive promoter pGAP (see BBa_K431009). pFUS1 activation is monitored by the production of RFP.

As a control, we firstly demonstrated the activity of the pGAP promotor (see BBa_K431009) used to constitutively express Odr-10.

We tested the functionality of the Odr-10 receptor by growing the cells on a media specifically designed to induce the activation of Ste proteins (http://2017.igem.org/Team:INSA-UPS_France/Protocols).

Absorbance and fluorescence production by P. pastoris strain having integrated the empty plasmid or the plasmid containing Odr-10/pFUS-RFP system was followed over the time in a microplate reader. Results are presented in Figure 2.

Figure 2: Measurement of pFUS1 activity. P. pastoris was grown in CMM media supplemented with glutamine and without diacetyl. Negative control (T-) was performed with P. pastoris with genomic integration of pPICZα, P. pastoris with pPICZα-ODR10/pFUS1-RFP genomic integration were grown with 500µM or 1000 µM of diacetyl. P. pastoris with genomic integration of pPICZα-pGAP-RFP (constitutive expression, T+) is the positive control. Results are presented as the ratio of RFP fluorescence at 600(+/- 10) nm divided by absorbance at 595 nm (measure of cell density). Results are issued from duplicated experiments.

No difference were observed between the control and the strain expressing Odr-10/pFUS1-RFP + 500 µM diacetyl. However, when diacetyl wads present at higher concentration (i.e. 1000µM), significant increase in expression was observed (reproduced twice).

This result demonstrated the functionality of the complete detection pathway activated by Odr-10 diacetyl detection.