Difference between revisions of "Part:BBa K1072023"

Revision as of 10:33, 11 October 2022

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 characterize pFUS1 activation. Indeed, when diacetyl binds to Odr-10 (BBa_K1072010) a cascade of activation of Ste proteins (endogenous to P. pastoris) leads to the binding of Ste12 on pFUS1 promoter, and so to an increase of transcription from pFUS1 promoter (revealed by the RFP reporter gene (BBa_J04450).

Figure 1: Construction to characterize pFUS1 promoter A diacetyl receptor (Odr-10) is expressed under the control of the constitutive 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 (http://2017.igem.org/Team:INSA-UPS_France/Protocols).

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 on 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-) was performed with P. pastoris having genomic integration of pPICZα, P. pastoris with pPICZα-ODR10/pFUS1-RFP genomic integration were grown with 500 µM or 1000 µM of diacetyl. P. pastoris with genomic integration of pPICZα-RFP (T+) is the positive control. Results are issued 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).

No difference was observed between the negative control and the strain expressing Odr-10/pFUS1-RFPwith 500 µM diacetyl. However, when diacetyl was present at higher concentration (i.e. 1000 µM), significant differences were observed between both strain. This data demonstrate the functionality of the complete detection pathway, including the pFUS1 functionality.

Team Estonia_TUIT characterization of BBa_K1072023 (pFUS1)

pFUS1 controls the expression of the FUS1gene that encodes a membrane protein from the yeast mating pathway. Fus1 protein localizes to the tip of the shmoo (a protrusion formed during yeast mating) (Lim et al., 2014). Fus1 protein coordinates the signaling, cell fusion, and polarization events required for the fusion of yeast cells. Transcription of FUS1 is mediated in haploid yeast by four pheromone response elements (PRE) of the pFUS1 promoter (Hagen et al., 1991). To induce the pFUS1, we used the α-factor to activate the mating pathway in MATa yeast strain.

pFUS1 promoter activity was induced with 1 μg/mL of α-factor. We observed gradual pheromone-dependent activation of the pFUS1 promoter (Fig. 1) throughout the experiment. When α-factor was not present (uninduced strain), the level of GFP fluorescence was similar to those observed in the control DOM090 strain (data not shown). Fus1 protein is associated with the tip of the shmoo involved in a mating process. Since the α-factor is not removed from the environment and mating did not happen, it leads to the constant activation of pFUS1 promoter and, as a result, an increase in GFP fluorescence throughout the experiment (Figure 1).

Figure 1. pFUS1-EGFP fluorescence intensity in time during α-factor exposure. The plot shows the mean fluorescence levels of a population of cells from cultures without α-factor (uninduced) or with α-factor (induced).

References:

• Lim, W., Mayer, B., & Pawson, T. (2014). Cell Signaling : Principles and mechanisms. Cell Signaling. https://doi.org/10.1201/9780429258893
• Lawrence, C. W. (2004). Cellular functions of DNA polymerase ζ and REV1 protein. Advances in Protein Chemistry, 69, 167–203. https://doi.org/10.1016/S0065-3233(04)69006-1