Difference between revisions of "Part:BBa K1072023"

Revision as of 12:12, 28 October 2017

Fus1 Promotor

BBa_K1072023 is a induced promotor, originating from S.cerevisiae.In the endogenous MAP-kinase pathway in S.cerevisiae, third messenger,ste12, will stimulate fus1 promoter, which results in mating of the yeast.However the sequence of fus1 contains Pst1 restrict enzyme site, we use the method of overlap extension to achieve the purpose of site-directed mutagenesis ("CTGCAG" to "CTGGAG").

This part is an improvement of BBa_K775004 of 2012 TU delft team.

Sequence and Features

Team INSA-UPS France 2017: usage of BBa_K1072023 in Pichia pastoris strain : activation of pFUS1 promoter upon diacetyl detection by Odr-10 receptor

We wanted to build a gene with a diacetyl inducible expression using Odr-10/pFUS1 system. To do so, we designed the construction (see figure 1) to characterised pFUS1 activation. Indeed, when diacetyl binds to Odr-10 (BBa_K1072010) a cascade of activation of Ste proteins (endogenous to P. pastoris) will lead to the binding of Ste12 on pFUS1 promoter, and so to the transcription of pFUS1 reporter gene. RFP(BBa_J04450) was used as gene rapporter.

Figure 1: Construction to characterized pFUS1 promoter A diacetyl receptor (Odr-10) is expressed under the control of a constitutuve promoter pGAP (BBa_K431009). pFUS1 activation is monitored by RFP reporter gene.

As a control, we firstly demonstrated the activity of the pGAP promotor (see BBa_K431009) present in the yeast vector pPICZα we used)

We tested the functionality of the complete system Odr-10/pFUS1 by growing the cells on a media specifically designed to induce the activation of Ste proteins ) and that contained diacetyl.

Absorbance and florescent 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 w/o diacetyl. Negative control (T-) is performed with P. pastoris with genomic integration of pPICZα, P. pastoris with pPICZα-ODR10/pFUS1-RFP genomic integration were grown with 500µM and 1000 µM of diacetyl. P. pastoris with genomic integration of pPICZα-RFP (T+) is the positive Results from duplicated experiments. Results are presented as the ratio of RFP fluorescence at 600(+/- 10) nm divided by absorbance at 595 nm (measure of cell density).

In the conditions w/o diacetyl or with 500µM of diacetyl no difference are observed between the control and the strain expressing Odr-10/pFUS1-RFP. However, when diacetyl is present at higher concentration (i.e. 1000µM), significant difference are observed between both strain.

The RPF fluorescence is higher in the P. pastoris strain having integrated the plasmid containing Odr-10/pFUS1-RFP system than the one having integrated the empty plasmid. This data demonstrate the functionality of the complete detection pathway to increase the activation of pFUS1. The RPF fluorescence is higher in the P. pastoris strain having integrated the plasmid containing Odr-10/pFUS1-RFP system than the one having integrated the empty plasmid. This data demonstrate the functionality of the complete detection pathway to increase the activation of pFUS1.