Composite

Part:BBa_K3610051

Designed by: Jonas Sebastian Trottmann   Group: iGEM20_UZurich   (2020-10-09)
Revision as of 21:54, 22 October 2020 by Jtrott (Talk | contribs) (→‎Luminescence assay)


CORE ectodomain / SmallBit NanoLuc for S. cerevisiae

This part includes the ectodomain of the plant pattern recognition receptor CORE fused to the SmallBit part of the split-NanoLuc system. To ensure localization at the membrane, this part further contains the sequence for the signal peptide of the alpha factor from S. cerevisiae.


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 additionally 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.

Usage with NanoLuc

In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.

The ligand-dependent interaction of CORE with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither CORE nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, csp22-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a luminescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs CORE and BAK1. This enables us to use this part, in coordination with the BAK1 ectodomain and LargeBit NanoLuc, to visually capture the presence of the csp22 epitope in water samples, as the csp22 pattern will induce interaction between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a functional protein, which gives a visual uotput with the substrate furimazine.


Characterization

We coexpressed this part together with Part:BBa_K3610038 (eBAK1), which is the ectodomain of the co-receptor BAK1 fused to the LargeBit part of the NanoBit system, in S. cerevisiae. The parts were assembled with Golden Gate Cloning in two different vectors. This part (eCORE) was assembled in a plasmid containing a kanamycin resistance gene, while the eBAK1 construct was assembled in a plasmid with a TRP1 gene, which encodes an enzyme necessary for tryptophan synthesis. These two genes allowed for selction on selective media.

After coexpressing the two plasmids in S. cerevisiae, a dimerization assay under a luminometer was performed with a plate reader of the type Synergy H1. Should both proteins be expressed and able to interact, then it is possible that functionality of the split-NanoLuc protein get reconstituted, which would enable it to catalyze the reaction of furimazine to furimamide, a reaction which is accompanied by luminescence.

Luminescence assay

Samples with cells which were transfected with the two mentioned plasmids (eBAK1 and eCORE) and samples containing S. cerevisiae cells that were not transfected with any plasmids (UT).

Optical densities (OD600) of all samples were adjusted to 0.34.
For each type of sample, three types of measurements were made:

  • 1 µL of deionized water added (no elicitor)
  • 1 µL of epitope elf18 added
  • 1 µL of epitope csp22 added

Each measurement was done four times with sample size 50µL. To each well 50 µL NanoGlo solution was added (50:1 buffer to furimazine)
The following table contains the results of the plate reader.

Time T° Lum UT UT UT UT UT + csp22 UT + csp22 Ut + csp22 UT + csp22 UT + elf18 UT + elf18 UT + elf18 UT + elf18 CORE CORE CORE CORE CORE + csp22 CORE + csp22 CORE + csp22 CORE + csp22 CORE + elf18 CORE + elf18 CORE + elf18 CORE + elf18
00:01:21 22.9 10 9 8 8 11 8 8 9 9 8 8 10 339 338 294 261 287 218 237 272 192 205 225 176
00:06:21 22.9 10 9 9 10 9 9 8 8 8 9 9 9 386 348 303 259 282 245 240 270 207 214 196 173
00:11:21 23 8 9 10 8 8 9 8 10 8 8 8 8 392 379 302 296 309 239 264 271 209 204 199 168
00:16:21 23 11 8 9 9 8 10 8 9 9 9 9 9 409 357 325 301 307 241 265 254 214 199 197 171
00:21:21 23.1 9 9 9 10 10 10 8 8 9 8 10 9 401 375 305 300 295 253 244 279 216 194 207 166
00:26:21 23.1 9 11 9 10 9 9 9 9 9 9 8 8 398 395 318 273 295 238 254 256 212 203 183 169
00:31:21 23.1 8 9 9 8 9 9 9 9 8 9 8 9 404 369 310 294 303 238 267 230 204 192 165 157
00:36:21 23.2 10 10 9 8 8 8 9 8 8 9 9 8 405 387 290 282 290 239 232 231 194 196 175 160
00:41:21 23.2 9 8 9 9 9 9 9 8 8 11 8 8 407 380 270 292 304 239 256 228 203 188 161 146
00:46:21 23.2 11 8 9 8 8 9 8 12 8 11 8 8 413 364 266 272 269 220 240 222 192 169 170 149
00:51:21 23.3 9 9 8 13 9 8 9 10 8 8 12 9 383 355 266 247 274 227 222 213 189 151 162 140
00:56:21 23.3 9 9 8 10 9 8 9 9 9 8 10 9 388 369 269 272 266 204 222 195 182 166 137 138
01:01:21 23.3 9 10 9 9 9 8 9 9 9 8 9 8 379 349 235 228 273 221 208 184 164 158 145 133
01:06:21 23.3 8 10 9 10 9 9 9 11 9 8 9 10 376 340 223 238 263 208 194 195 176 152 140 121
01:11:21 23.3 9 8 9 8 8 10 8 9 10 9 8 9 346 339 238 216 242 211 190 175 162 138 131 119
01:16:21 23.3 9 9 8 9 9 9 9 9 11 10 9 9 345 345 189 218 247 199 189 165 149 127 135 133
01:21:21 23.3 8 8 10 10 9 10 10 8 9 9 8 10 345 319 203 214 250 195 188 169 148 148 127 127
01:26:21 23.3 9 9 9 9 9 10 10 9 9 10 10 10 347 326 199 212 244 189 189 159 159 128 136 121
01:31:21 23.3 9 9 9 9 9 8 9 9 9 11 9 9 326 309 188 216 225 187 180 157 140 117 125 113
01:36:21 23.3 9 9 9 8 9 9 9 9 9 8 8 8 347 295 170 197 224 180 168 157 148 114 109 100
01:41:21 23.3 9 10 10 9 10 9 8 9 9 9 10 10 313 298 178 186 225 189 159 148 133 117 123 97
01:46:21 23.3 9 8 9 10 8 8 10 8 9 10 10 11 302 293 160 181 220 170 167 144 130 120 113 109
01:51:21 23.3 11 10 9 9 9 9 9 8 9 10 8 9 296 283 164 160 216 172 149 145 119 98 113 106
01:56:21 23.3 9 8 8 10 9 8 9 10 9 10 10 10 283 257 147 161 194 159 139 138 123 108 115 108
02:01:21 23.3 8 9 9 9 9 9 9 10 9 11 9 9 282 264 150 169 178 162 141 133 119 94 118 102

The average values of the four measurements are displayed in the plot below.

Figure 1: Average Luminescence over time

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]


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