Part:BBa_K3610046

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.

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

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]