Difference between revisions of "Part:BBa K319003"

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===Characterization ===
 
===Characterization ===
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To visualize the expression level of [https://parts.igem.org/Part:BBa_K431009 pGAP] and choose the appropriate promoter for NLS-Csy4 and function genes, we measure the fluorescence intensity, and the data was divided by OD.
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To visualize the expression level of [https://parts.igem.org/Part:BBa_K3544011 pTEF1] and choose the appropriate promoter for NLS-Csy4 and function genes, we measure the fluorescence intensity, and the data was divided by OD.
  
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[[file:T--SCU-China--scu-2020-results-15.png|500px|center|]]
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[[image:T--SCU-China--scu-2020-results-15.png|600px|center|]]
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                  Figure 1: Relative fluorescence intensity of two fluorescence proteins under two promoters.
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Figure 1: Relative fluorescence intensity of two fluorescence proteins under two promoters.
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The signal of [https://parts.igem.org/Part:BBa_K3544204 pGAP-CRISPR(Csy4)-DsRed-tADH1] is low, this is related to our plasmid design. We use double enzyme digestion and T4 ligation to construct pGAP-NLS-yeGFP-28nt-DsRed, the first step is the construction of pGAP-28nt-DsRed, which is called pGAP-R here. The secondary structure of 28nt may influence the expression level of DsRed.
 
The signal of [https://parts.igem.org/Part:BBa_K3544204 pGAP-CRISPR(Csy4)-DsRed-tADH1] is low, this is related to our plasmid design. We use double enzyme digestion and T4 ligation to construct pGAP-NLS-yeGFP-28nt-DsRed, the first step is the construction of pGAP-28nt-DsRed, which is called pGAP-R here. The secondary structure of 28nt may influence the expression level of DsRed.
  
 
In short, pTEF1 is stronger than pGAP (judged by the fluorescence of GFP and DsRed), so we use pTEF1 to express NLS-Csy4 for high cutting efficiency, and to design the construction of function genes additionally according to our modeling result.
 
In short, pTEF1 is stronger than pGAP (judged by the fluorescence of GFP and DsRed), so we use pTEF1 to express NLS-Csy4 for high cutting efficiency, and to design the construction of function genes additionally according to our modeling result.

Latest revision as of 16:30, 27 October 2020


yeast TEF1 promoter

This is the full length TEF1 promoter including the distal and proximal portions and their regulatory elements.


Contribution

  • Group: [http://2016.igem.org/Team:Chalmers_Gothenburg iGEM Team Chalmers Gothenburg 2016]
  • Author: John Hellgren
  • Summary: A promoter study to characterize this promoter and compare it against several others in two different conditions.

Characterization

A promoter study was performed to characterize this promoter. The reporter gene GFP was cloned into the replicative plasmid p416tef, downstream of the TEF1 promoter. 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 pAQR1, pGLN1, pPCK1 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 [1]. 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].

Contribution

  • Group: [http://2018.igem.org/Team:Peking Peking iGEM team 2018]
  • Author: Chen Yuyang
  • Summary: characterize the strength of this promoter and compare it with other promoters.
  • Link to our biobrick: https://parts.igem.org/Part:BBa_K2601037

Characterization

We have tested the strength of TEF1 using flow cytometry and compared it with other promoters. The promoters are fused with Frb-HOTag6 and FKBP-HOTag3 respectively.

T--Peking--promoter-strength-new.png

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 205


References

[1] K. Weinhandl, M. Winkler, A. Glieder, and A. Camattari, “Carbon source dependent promoters in yeasts,” Microbial Cell Factories, vol. 13, no. 1, 2014

Contribution

Characterization

To visualize the expression level of pTEF1 and choose the appropriate promoter for NLS-Csy4 and function genes, we measure the fluorescence intensity, and the data was divided by OD.

T--SCU-China--scu-2020-results-15.png
                  Figure 1: Relative fluorescence intensity of two fluorescence proteins under two promoters.

The signal of pGAP-CRISPR(Csy4)-DsRed-tADH1 is low, this is related to our plasmid design. We use double enzyme digestion and T4 ligation to construct pGAP-NLS-yeGFP-28nt-DsRed, the first step is the construction of pGAP-28nt-DsRed, which is called pGAP-R here. The secondary structure of 28nt may influence the expression level of DsRed.

In short, pTEF1 is stronger than pGAP (judged by the fluorescence of GFP and DsRed), so we use pTEF1 to express NLS-Csy4 for high cutting efficiency, and to design the construction of function genes additionally according to our modeling result.