Difference between revisions of "Part:BBa K3610046"
(→‎Characterization)
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Imaging did not reveal increased fluorescence at the excitation and emission wavelengths for YFP.
 
Imaging did not reveal increased fluorescence at the excitation and emission wavelengths for YFP.
 +
 +
====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 <i>S. cerevisiae</i> 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.
 +
 +
{| class="wikitable" width="95%"
 +
|-
 +
! scope="col", colspan ="10"| measured OD600 values (OD)
 +
|-
 +
! scope="col"|
 +
! scope="col", colspan="3"| Replicate 1
 +
! scope="col", colspan="3"| Replicate 2
 +
! scope="col", colspan="3"| Replicate 3
 +
|-
 +
! scope="row"| 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
 +
|}
 +
 +
{| class="wikitable" width="95%"
 +
|-
 +
! scope="col", colspan ="10"| Fluorescence Top Reading (FTR)
 +
|-
 +
! scope="col"|
 +
! scope="col", colspan="3"| Replicate 1
 +
! scope="col", colspan="3"| Replicate 2
 +
! scope="col", colspan="3"| Replicate 3
 +
|-
 +
! scope="row"| 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 <i>blank corrected</i> (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.
 +
{| class="wikitable"
 +
|-
 +
!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.
 +
 +
{| class="wikitable"
 +
|-
 +
!Control
 +
!eCORE
 +
!eEFR
 +
!BAK-
 +
!BAK+
 +
!eBAK
 +
|-
 +
|1||0,8871565975||5,586286516||6,228390841||2,325233033||6,718461693
 +
|}
 +
 +
<br>
 +
[[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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Revision as of 07:20, 21 October 2020


CORE ectodomain / YFP

This part contains the ectodomain of the plant cell surface receptor CORE from S. lycopersicum 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

CORE

The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.

CORE 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 CORE ectodomain / YFP in S. cerevisiae

After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope.

T--UZurich--eCORE.png

Imaging did not reveal increased fluorescence at the excitation and emission wavelengths for YFP.

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).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1740
    Illegal BamHI site found at 373
    Illegal BamHI site found at 1765
    Illegal BamHI site found at 2117
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]