Part:BBa_K3610045

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.

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

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]