Difference between revisions of "Part:BBa K3610045"

(Spectrometry)
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!Control
 
!Control
!BAK+
 
!BAK-
 
!eBAK
 
 
!eCORE
 
!eCORE
 
!eEFR
 
!eEFR
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!BAK-
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!BAK+
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!eBAK
 
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|1||0,8871565975||5,586286516||6,228390841||2,325233033||6,718461693
 
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[[File:T--UZurich--Spectrometer_1.png|500px|none|left|Figure 3: Fluorescence values standardized for OD600 of the different receptors (C=Control). Cells with BAK+ showed only weak fluorescence, while BAK-, eBAK and eEFR showed a strong increase in the fluorescence levels. CORE did not display any increase when compared with untreated <i>S. cerevisiae</i> cells (autofluorescence).]]
 
[[File:T--UZurich--Spectrometer_1.png|500px|none|left|Figure 3: Fluorescence values standardized for OD600 of the different receptors (C=Control). Cells with BAK+ showed only weak fluorescence, while BAK-, eBAK and eEFR showed a strong increase in the fluorescence levels. CORE did not display any increase when compared with untreated <i>S. cerevisiae</i> cells (autofluorescence).]]
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Results of the plate reader suggested increased fluorescence through expression of YFP. The same was observed when cells were examined with the microscope. Therefore, <i>S. cerevisiae</i> cells express the plasmid containing the EFR ectodomain. Additionally, microscopy revealed localization at the cell membrane. This localization at the cell periphery is a big success as it shows, that the signal peptide from the alpha-Mating factor did, in fact induce translation into the membrane. Additionally, it was shown that the intracellular kinase domain is not necessary for membrane localization.
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This expression of the EFR ectodomain in <i> S. cerevisiae</i>, which is accompanied by localization at the cell membrane is a big achievement that opens the doors for many future applications.
  
 
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Revision as of 07:14, 21 October 2020


EFR ectodomain / YFP

This part contains the ectodomain of the plant cell surface receptor EFR from A. thaliana fused to a yellow fluorescent protein. This part lacks the natural N-terminal signal sequence but instead uses the signal sequence from the alpha-Factor from yeast.

Usage and Biology

EFR

Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants first defence mechanism against potential pathogens. The EFR receptor is also a leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.

EFR with YFP

In this sequence, the C-terminal domain entailing the intracellular kinase domain was replaced with the sequence coding for the yellow fluorescent protein venus, while the ectodomain and the transmembrane domain, including the juxtamembrane domain were kept. Additionally, a signal sequence native to S. cerevisiae was fused to the N-terminal sequence, which does not contain the native signal peptide. This way, the protein can be integrated into the membrane during translation and the expression can be observed as with the receptor protein, the YFP (Exλ : 515 nm, Emλ : 528 nm) gets translated as well.

Characterization

Expression of EFR ectodomain/YFP in S. cerevisiae

In a first step we inserted the single fragments making up this part into a plasmid with a gentamycin-3-acetyltransferase gene and transformed E. coli (DH10alpha) with the plasmids for amplification. In the next step we assembled the fragments in a plasmid with a spectinomycin acetyltransferase and amplified the plasmids again in the same E. coli strain. For this step we applied the techniques of Golden Gate Cloning to get the fragments in the right order into the plasmid. The restriction enzyme we chose was BsaI. For expressing this part consisting of YFP and the receptor protein, we initially intended to use promoters of different strength to get more quantitative data. Finally, we got the construct in a plasmid with a truncated version of the ADH1 promoter from S. cerevisiae. For termination, this part has the terminator sequence of the enolase 2 protein from S. cerevisiae. The plasmid also contained the TRP1 gene, which encodes phosphoribosylanthranilate isomerase, an enzyme that catalyzes the third step in tryptophan biosynthesis. This enabled us to use the same plasmid for expression in S. cerevisiae. We prepared a medium containing YNB and free amino acids, without tryptophan. S. cerevisiae cells (AP4) were transfected with the plasmid and then plated on the selective medium.

Microscopy

After successful transformation of yeast cells we checked for enhanced fluorescence with confocal fluorescence microscopy.

T--UZurich--eEFR.png T--UZurich--Control.png


Imaging of the S. cerevisiae cells, which had been previously transfected with plasmids containing this construct, revealed increased fluorescence levels than the untransfected control. These results imply increased expression of YFP, indicating expression of the EFR ectodomain. Additionally, the imaging results suggest that the proteins are in part localized at the cellular membrane, which is in alignment with our expectations as there is a secretion signal peptide and a transmembrane domain in the construct.

Spectrometry

In addition to analyzing the cells with a microscope, we conducted a fluorescence assay with a plate reader. We conducted this experiment for multiple receptors at the same time. This way we were able to compare the expression levels of differnt versions of the BAK1 receptor. For each receptor we tried to isolate three different biological samples, however, not all cells grew. Ultimately, we only had two samples for the following S. cerevisiae cells: untransformed (Control), transformed with BAK1 ectodomain fused to YFP (eBAK) and the CORE ectodomain fused to YFP (eCORE). For the BAK1 with and without the native signal peptide fused to YFP (BAK+ and BAK-) and the EFR ectodomain fused to YFP (eEFR), we had samples from three different colonies. For each biological replicate, the optical density at absorbance of 600 nm (OD600) and the fluorescence levels were measured three times.

measured OD600 values (OD)
Replicate 1 Replicate 2 Replicate 3
Blank 0,08200000226 0,08200000226 0,08389999717
Control 0,3806000054 0,3747999966 0,4221999943 0,1316999942 0,131400004 0,1176000014
BAK+ 0,4943000078 0,4638999999 0,4514000118 0,5781000257 0,5253999829 0,5799999833 0,2615999877 0,2171999961 0,2011999935
BAK- 1,417099953 1,365499973 1,368899941 0,6305999756 0,5633999705 0,6216999888 0,896600008 0,7882999778 0,8032000065
eBAK 1,009699941 0,8404999971 0,8934999704 0,2653000057 0,2368000001 0,2592999935
eCORE 1,021499991 0,8616999984 0,9178000093 0,826300025 0,6888999939 0,7401999831
eEFR 1,379699945 1,322700024 1,333500028 1,035899997 1,014000058 0,9526000023 0,4860999882 0,3797000051 0,3829999864

The following settings were applied for fluorescence measurements:

Mode: Fluorescence Top Reading
Excitation Wavelength: 485 nm
Emission Wavelength: 535 nm
Excitation Bandwidth: 20 nm
Emission Bandwidth: 25 nm
Temperature: 22.3°C
Fluorescence Top Reading (FTR)
Replicate 1 Replicate 2 Replicate 3
Blank 1297 1282 1322
Control 2684 2474 2634 1852 1792 1750
BAK+ 3038 2813 2760 2836 2493 2788 2084 2072 2067
BAK- 35794 30319 31424 10792 9097 10517 22609 20227 21220
eBAK 26455 19828 21613 6614 5507 6229
eCORE 10709 8382 9339 8957 7062 7735
eEFR 43125 37782 39589 25641 24668 22517 12410 9054 9027

After measurement of the optical density and the fluorescence, the data were blank corrected (the average of the three blank measurements was subtracted from each measurement value).

The average of each of the three (or two) samples was calculated. From these values, the average was taken again.

After this step, we normalized the fluorescent output for OD600 (FTR/OD). The results of these calculations are displayed in the table below.

Control BAK+ BAK- eBAK eCORE eEFR
4185,221063 9731,614266 26067,19254 28118,24739 3712,946478 23379,84399

If we set the values for the Control to 1 (Control = 1), then we get the fluorescence levels relative to the control, which is again diplayed in the table below.

Control eCORE eEFR BAK- BAK+ eBAK
1 0,8871565975 5,586286516 6,228390841 2,325233033 6,718461693


Figure 3: Fluorescence values standardized for OD600 of the different receptors (C=Control). Cells with BAK+ showed only weak fluorescence, while BAK-, eBAK and eEFR showed a strong increase in the fluorescence levels. CORE did not display any increase when compared with untreated S. cerevisiae cells (autofluorescence).

Results of the plate reader suggested increased fluorescence through expression of YFP. The same was observed when cells were examined with the microscope. Therefore, S. cerevisiae cells express the plasmid containing the EFR ectodomain. Additionally, microscopy revealed localization at the cell membrane. This localization at the cell periphery is a big success as it shows, that the signal peptide from the alpha-Mating factor did, in fact induce translation into the membrane. Additionally, it was shown that the intracellular kinase domain is not necessary for membrane localization.

This expression of the EFR ectodomain in S. cerevisiae, which is accompanied by localization at the cell membrane is a big achievement that opens the doors for many future applications.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 355
    Illegal NheI site found at 1285
    Illegal NheI site found at 2203
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1871
  • 1000
    COMPATIBLE WITH RFC[1000]