Composite

Part:BBa_K3610032

Designed by: Jonas Sebastian Trottmann   Group: iGEM20_UZurich   (2020-10-05)
Revision as of 22:53, 20 October 2020 by Jtrott (Talk | contribs) (→‎Spectrometer)


BAK1 ectodomain / YFP

This part contains the ectodomain of the plant cell surface receptor 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 S. cerevisiae.

Usage and Biology

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucine-rich repeat receptor kinase (LRR-RK) which interacts with multiple other LRR-RKs with different functions in hormone signalling and defense response. BAK1 localizes at the plasma membrane and the endosome. The BAK1 protein forms a structure with an extracellular domain with leucine-rich repeats, a single pass transmembrane domain and an intracellular domain with a kinase function.

Among others, BAK1 interacts with the LRR-RKs EF-Tu receptor (EFR), Flagellin sensing 2 (FLS2) and cold-shock protein receptor (CORE), all of which are pathogen recognition receptors (PRR) in brassicaceae plants. Upon binding of a microbe-associated molecular pattern at the LRR domain of the PRR, BAK1 forms a heterodimer with the PRR which triggers a phosphorylation cascade, leading to upregulation of defense mechanisms.

BAK1 fused to 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. Additionally, the YFP (Exλ : 515 nm, Emλ : 528 nm) gets translated together with the receptor protein, which allows observation of expression and localization under a microscope and measurement of the strength of the expression with a fluorometer.

Characterization

Expression of BAK1 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.

Fluorescent Microscopy

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

T--UZurich--eBAK.png T--UZurich--Control.png


Confocal microscopy confirmed increased fluorescence in the S. cerevisiae cells that had been previously transfected with plasmids containing BAK1 ectodomain fused to YFP. This increased fluorescence indicates expression of our genes. Additionally, this imaging experiment revealed that the fluorescent protein is in part localized at the cell periphery. This is in alignment with our expectations as our construct includes a secretion signal protein and a receptor coding protein with the transmembrane domain. These results suggest that the secretion peptide fused to the receptor ectodomain, including the transmembrane domain can be expressed in S. cerevisiae and that the components are sufficient for localization at the cell membrane.

Spectrometer

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 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
Fluorescence (YFP)
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 (YFP/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 BAK+ BAK- eBAK eCORE eEFR
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
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 867
    Illegal PstI site found at 912
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 867
    Illegal PstI site found at 912
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 867
    Illegal PstI site found at 912
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 867
    Illegal PstI site found at 912
  • 1000
    COMPATIBLE WITH RFC[1000]


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