Difference between revisions of "Part:BBa K1072023"

Line 44: Line 44:
  
 
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.
 
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 FUS1 gene 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 <i>et al</i>., 2014). Fus1 protein coordinates the signaling, cell fusion and polarization events required for the fusion of yeast cells. Transcription of <i>FUS1</i> is mediated in haploid yeast by four pheromone response elements (PRE) of the pFUS1 promoter (Hagen <i>et al</i>., 1991). To induce the pFUS1, we used the α-factor to activate the mating pathway in MATa yeast strain.
 +
 +
We quantified the expression driven by the FUS1 promoter using fluorescent proteins as reporter genes. The expression cassettes were integrated into the yeast genome, and the fluorescence of the reporter proteins was monitored by quantitative time-lapse microscopy. The fluorescence levels were compared to the cell background fluorescence of the control DOM90 strain, which does not express any fluorescent proteins (Fig. 1).
 +
 +
<i>FUS1</i> promoter activity was induced with 1 μg/mL of α-factor. We observed strong pheromone-dependent activation of the <i>FUS1</i> promoter (Fig. 1, 2).
 +
 +
[[Image:Estonia TUIT pFUS1 bar.png|600px|thumb|center|<b>Figure 1.</b> The fluorescence intensity of EGFP expressed from the pFUS1 promoter in the presence (induced) or absence (uninduced) of α-factor. Bars indicate the mean EGFP fluorescence intensities of a population of cells from the indicated conditions six hours after α-factor addition. Error bars show standard deviation. 
 +
]]
 +
 +
[[Image:Estonia TUIT pFUS1 line.png|600px|thumb|center|<b>Figure 2.</b> 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 (un-induced) or with α-factor (induced).]]
 +
 +
<b>References:</b>
 +
<ul>
 +
<li>Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. <i>ACS Synthetic Biology, 4</i>(9), 975–986. https://doi.org/10.1021/SB500366V/SUPPL_FILE/SB500366V_SI_002.ZIP</li> 
 +
<li>Lawrence, C. W. (2004). Cellular functions of DNA polymerase ζ and REV1 protein. <i>Advances in Protein Chemistry, 69, </i>167–203. https://doi.org/10.1016/S0065-3233(04)69006-1</li>
 +
</ul>

Revision as of 04:38, 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


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_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 FUS1 gene 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.

We quantified the expression driven by the FUS1 promoter using fluorescent proteins as reporter genes. The expression cassettes were integrated into the yeast genome, and the fluorescence of the reporter proteins was monitored by quantitative time-lapse microscopy. The fluorescence levels were compared to the cell background fluorescence of the control DOM90 strain, which does not express any fluorescent proteins (Fig. 1).

FUS1 promoter activity was induced with 1 μg/mL of α-factor. We observed strong pheromone-dependent activation of the FUS1 promoter (Fig. 1, 2).

Figure 1. The fluorescence intensity of EGFP expressed from the pFUS1 promoter in the presence (induced) or absence (uninduced) of α-factor. Bars indicate the mean EGFP fluorescence intensities of a population of cells from the indicated conditions six hours after α-factor addition. Error bars show standard deviation.
Figure 2. 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 (un-induced) or with α-factor (induced).

References: