Difference between revisions of "Part:BBa K2030000"
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<partinfo>BBa_K2030000 short</partinfo>
 
<partinfo>BBa_K2030000 short</partinfo>
  
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The upstream regulatory sequence to the gene <i>AQR1</i>, coding for a transporter that facilitates secretion of excess amino acids. It is induced by amino acids [1].
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The upstream regulatory sequence to the gene <i>AQR1</i> from <i>Saccharomyces cerevisiae</i> CEN.PK113-5D, coding for a transporter that facilitates secretion of excess amino acids. It is induced by amino acids [1].
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===Characterization===
 
===Characterization===
 
A promoter study was performed to characterize this promoter. The <i>AQR1</i> promoter was cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. By using a replicative plasmid instead of chromosomal integration, a higher copy number can be achieved, which will make sure that even weak promoters give a detectable signal. For the glucose conditions, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, diluted to OD<SUB>600</SUB>=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured in a 96-well plates (NUNC 96) in a BMG Labtech FLUOstar Omega plate reader with triplicate samples using the following setting: 20 flashes per well, excitation/emission wavelength at 485/520 nm and gain set to 800.  
 
A promoter study was performed to characterize this promoter. The <i>AQR1</i> promoter was cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. By using a replicative plasmid instead of chromosomal integration, a higher copy number can be achieved, which will make sure that even weak promoters give a detectable signal. For the glucose conditions, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, diluted to OD<SUB>600</SUB>=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured in a 96-well plates (NUNC 96) in a BMG Labtech FLUOstar Omega plate reader with triplicate samples using the following setting: 20 flashes per well, excitation/emission wavelength at 485/520 nm and gain set to 800.  
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[[Image:T--Chalmers Gothenburg--glucose-acetate-relative.png|800px|thumb|center|Figure 1: Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Triplicate runs were made for each promoter and error bars are shown as confidence intervals with p=0.05, using student's t-test.]]
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[[Image:T--Chalmers Gothenburg--glucose-acetate-relative.png|800px|thumb|center|Figure 1: Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Each sample was loaded into three different wells in the plate reader, and error bars are shown as confidence intervals with p = 0.05, using student's t-test.]]
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All promoters except pPCK1 show higher expression relative pTEF1 at glucose conditions compared with acetate conditions, which is consistent with previous reports [2]. pPCK1 even has higher expression level than pTEF1, which means that pPCK1 could be preferred for overexpression when acetate is the only carbon source.
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A more detailed version of the promoter study and how it's connected to our project can be found [http://2016.igem.org/Team:Chalmers_Gothenburg/Project/Promoter_study here].
  
 
===Uploads===
 
===Uploads===
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===References===
 
===References===
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[1]: Velasco, I., Tenreiro, S., Calderon, I.L. and André, B., 2004. Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids. Eukaryotic Cell, 3(6), pp.1492-1503.
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[1] Velasco, I., Tenreiro, S., Calderon, I.L. and André, B., 2004. Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids. Eukaryotic Cell, 3(6), pp.1492-1503.<br>
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[2] K. Weinhandl, M. Winkler, A. Glieder, and A. Camattari, “Carbon source dependent promoters in yeasts,” <i>Microbial Cell Factories</i>, vol. 13, no. 1, 2014.

Latest revision as of 16:56, 19 October 2016

pAQR1 S. cerevisiae promoter

The upstream regulatory sequence to the gene AQR1 from Saccharomyces cerevisiae CEN.PK113-5D, coding for a transporter that facilitates secretion of excess amino acids. It is induced by amino acids [1].

Characterization

A promoter study was performed to characterize this promoter. The AQR1 promoter was cloned into the replicative plasmid p416tef by replacing the existing pTEF1 promoter and adding GFP as a reporter gene. By using a replicative plasmid instead of chromosomal integration, a higher copy number can be achieved, which will make sure that even weak promoters give a detectable signal. For the glucose conditions, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, diluted to OD600=0.3 in the same media and cultivated for 3 hours. The expression of GFP was measured in a 96-well plates (NUNC 96) in a BMG Labtech FLUOstar Omega plate reader with triplicate samples using the following setting: 20 flashes per well, excitation/emission wavelength at 485/520 nm and gain set to 800.

The cells were also grown in SD -URA + 0.5 % acetate to compare the expression levels when acetate was the only carbon source, which is connected to our coculture project. For the acetate experiment, the cells were grown as a preculture in SD -URA + 2 % glucose media overnight, washed and diluted to OD600=0.3 in SD -URA + 0.5 % acetate and cultivated for 24 hours before plate reader measurements. The longer cultivation time was due to slow growth with acetate as the carbon source. Furthermore, the reason for the longer cultivation time was to make sure that the GFP produced during the preculture in glucose was degraded.

The experiment was also done with the promoters pGLN1, pPCK1, pPYK2 and pTEF1 in the same way, and the results compared against each other. The raw data from the promoter study was normalized against OD600 of that sample, and the mean value of the negative control (cells with p416tef without GFP) was subtracted. The results are shown in Table 1.


Table 1. Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1,
PYK2 and pTEF1 for cells cultivated in SD -URA media + 2 % glucose or 0.5 % acetate (n=3).

Promoter Condition
Glucose (fluorescent unit/OD600)
 Acetate (fluorescent unit/OD600)
pAQR1
 303 63
pGLN1
 862 426
pPCK1 235 1721
pPYK2 125 77
pTEF1 1314 1399


In Figure 1 the results are normalized against the expression level of the pTEF1 promoter.


Figure 1: Fluorescent levels of GFP under the control of the promoters pAQR1, pGLN1, pPCK1, PYK2 and pTEF1 in glucose and acetate conditions relative the levels of pTEF1. Each sample was loaded into three different wells in the plate reader, and error bars are shown as confidence intervals with p = 0.05, using student's t-test.

All promoters except pPCK1 show higher expression relative pTEF1 at glucose conditions compared with acetate conditions, which is consistent with previous reports [2]. pPCK1 even has higher expression level than pTEF1, which means that pPCK1 could be preferred for overexpression when acetate is the only carbon source.

A more detailed version of the promoter study and how it's connected to our project can be found [http://2016.igem.org/Team:Chalmers_Gothenburg/Project/Promoter_study here].

Uploads

Promoter study data

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]


References

[1] Velasco, I., Tenreiro, S., Calderon, I.L. and André, B., 2004. Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids. Eukaryotic Cell, 3(6), pp.1492-1503.
[2] K. Weinhandl, M. Winkler, A. Glieder, and A. Camattari, “Carbon source dependent promoters in yeasts,” Microbial Cell Factories, vol. 13, no. 1, 2014.