Part:BBa_K3610032

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.

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 strengths 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. If expression of YFP (λEx = 515 nm, λEx = 528 nm) can clearly be observed, it is reasonable to assume that the receptor domain is expressed as well, as the YFP is fused to the receptor protein. Expression of the construct was confirmed. We failed, however, to confirm localization at the cell membrane.

Figure 1: Confocal microscopy of the normal S. cerevisiae cells (control).

Figure 2: Confocal microscopy of S. cerevisiae cells transformed with plasmids containing the BAK1 ectodomain fused to YFP. The results imply expression of the construct and also localization at the cell membrane.

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. As localization at the cell membrane was something we were particularily interested in, we repeated the confocal microscopy step with an additional membrane stain. The cell membrane was stained with fm4-64, which fluoresces strongly after binding to the cell membrane ((λEX = 515nm and λEM = 640nm). The binding of the dye is happening rapidly and it is also reversible. If the time spent between staining and imaging is too long, then the dye will be taken up by the organism and stored in the vacuole. Imaging with a confocal microscope for YFP and the fm4-64 stain shows the spatial overlap of the red fluorescence of the stain and the yellow fluorescence of the protein fused to the receptors.

Figure 3: Untransformed Control,(A) : YFP, (B) : FM4-64, (C): light field. (D): merge.Imaging of untransfected S. cerevisiae cells reveals hardly any fluorescence within the YFP spectrum

Figure 4: eBAK S. cerevisiae: (A) : YFP, (B) : FM4-64, (C) : light field. (D): merge.Most eBAK cells show strong yellow fluorescence with only diffuse structures. About 1% of the cells, clear co-localization with FM4-64 in a ring structure indicate integration into the PM.

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 5: 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 from the plate reader and from confocal microscopy were in alignment. Measurement with the fluorometer suggested increased expression of YFP when S. cerevisiae cells are transfected with a plasmid that contains this part. These results together strongly imply, that the ectodomain of the plant PRR BAK1 from A. thaliana, which has been fused to the secretion signal from the alpha-Factor, is expressed in yeast and also gets localized at the cell membrane.

Flow Cytometry

It has been important to us to examine a sample with different approaches simultaneously, which is why we were eager to also measure fluorescence intensity by flow cytometry. In a first phase, 100,000 cells were measured from each biological replicate (488/530 FITC channel in a BD FACSCanto II flow cytometer). In the next phase, the biological replicates for each construct were pooled together and 200,000 cells from each sample were measured.

Figure 6: Left: single biological replicates. right: pooled samples. With the exception of the construct with eCORE, cells transfected with our constructs showed considerably higher overall fluorescence intensities than the negative control.

Flow catometry provided further evidence for expression of the construct in S. cerevisiae. Cells transfected with plasmids containing BAK- showed significantly increased fluorescence intensities when examined as single biological replicates, as well as when the replicates were pooled together in one sample.

During our project, expression of the ectodomain of BAK1 fused to YFP was confirmed with three different approaches. We used a fluorometric plate reader, which suggested a strong increase in fluorescence intesity and we also got similar results when measuring fluorescence with flow cytometry. In addition to these results, fluorescence imaging also showed increased fluorescence and about 1% of the cells showed a very clear ring structure which co-localized with the memrbane stain FM4-64. These results suggest that the receptor ectodomain can get expressed in S. cerevisiae and also it can get localized at the cell periphery, although a lot of the proteins get stuck and are localized within the cell.

These results are very promising and open many doors to future applications of this plant PRR in synthetic biology as they strongly suggest that expression of this receptor ectodomain and membrane localization (with the alpha-Factor signal peptide) can be achieved.

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]