Composite

Part:BBa_K3610038

Designed by: Jonas Sebastian Trottmann   Group: iGEM20_UZurich   (2020-10-05)


BAK1 ectodomain / LargeBit for S. cerevisiae

This part contains the sequence for the ectodomain of the plant surface receptor BAK1 from A. thaliana fused to the LargeBit of the split NanoLuc system. Additionally, instead of the signal peptide native to the plant receptor, there is the secretion signal of the alpha factor from yeast at the N-terminal domain of the receptor, replacing the original signal sequence.

Usage and Biology

BAK1

The BRI1-associated receptor kinase (BAK1) is a leucin-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 leucin-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.

Usage with split-NanoLuc

In this case, the C-terminal domain of BAK1, entailing the intracellular kinase domain, was removed from the sequence. Instead, the LargeBit part of the split-NanoLuc protein domain was fused to the C-terminal domain via a 15 amino acid linker.

Interaction between the BAK1, which is a coreceptor to many other PRRs, 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. Coexpressed with, for example, Part:BBa_K3610043, which is the PRR EFR that contains the N-terminal domain of the split-mCherry protein instead of the intracellular kinase domain, elf18-induced interaction between BAK1 and EFR is driving the reassembly of the LargeBit and the SmallBit of the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxygen, yielding furimamide and a fluorescent output. During our project, we expressed this part in S. cerevisiae in an attempt to visualize the ligand-dependent interaction between BAK1 and the respective plant PRR. This enables us to use this part, in coordination with different PRRs, to test for the presence of the epitopes which are recognized by the plant receptors.

Characterization

In our iGEM project, we designed a system to sense the presence of bacterial epitopes in water using this part.

We assembled this part with Golden Gate Cloning in a plasmid with a spectinomycin acetyltransferase, a selection marker, and amplified the plasmids in E. coli. The plasmids also contained the TRP1 gene. This encodes phosphoribosylanthranilate isomerase, an enzyme necessary for tryptophan synthesis. This made the same plasmids also suitable for expression in S. cerevisiae. We created plasmids containing this part with different promoters and we also transformed yeast cells with the different types of our plasmid. We had multiple versions of the ADH1 promoter from S. cerevisiae, one version was the full length promoter and the other one a truncated version.

This part was coexpressed in our yeast cells together with different parts, the parts Part:BBa_K3610043 and Part:BBa_K3610051, which encode for the EFR and the CORE receptor ectodomain respectively, with the ectodomains fused to the SmallBit part of the NanoBit system. These plasmids were obtained in the same manner, with the only difference that the selction marker for S. cerevisiae cells was not TRP1 but a gene for Kanamycin acetyltransferase.

After coexpressing the BAK1 ectodomain with either EFR of CORE in our yeast cells, a dimerization assay under a fluorometer was performed with a plate reader of the type Synergy H1.

Dimerization Assay

A dimerization assay was performed with samples of S. cerevisiae cells which were transfected with the following receptors (receptor ectodomains fused to the respective NanoBit part are referred to as: eBAK1, eEFR and eCORE):

  • Untransformed (UT)
  • eBAK1 + eEFR (EFR)
  • eBAK1 + eCORE (CORE)

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). After addition of the substrate furimazine and the bacterial elicitors were added to the samples and then incubated for 30 minutes. After this 30 minute incubation time, the measurement started.
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 EFR EFR EFR EFR EFR + csp22 EFR + csp22 EFR + csp22 EFR + csp22 EFR + elf18 EFR + elf18 EFR + elf18 EFR + 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 1279 1438 1524 1217 1160 1012 1071 1023 898 821 735 680
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 1258 1468 1409 1193 1079 955 958 973 931 840 739 718
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 1247 1369 1359 1160 992 869 932 933 956 914 814 760
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 1140 1314 1345 1120 979 860 1011 1010 969 901 853 801
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 1060 1234 1247 1072 993 871 1015 1037 955 889 806 812
00:26:21 23.1 9 11 9 10 9 9 9 9 9 9 8 8 398 395 318 273 295 238 254 256 212 203 183 169 997 1202 1199 1007 985 877 986 1003 911 876 827 821
00:31:21 23.1 8 9 9 8 9 9 9 9 8 9 8 9 404 369 310 294 303 238 267 230 204 192 165 157 964 1098 1077 943 988 843 970 978 865 859 808 824
00:36:21 23.2 10 10 9 8 8 8 9 8 8 9 9 8 405 387 290 282 290 239 232 231 194 196 175 160 906 1071 1061 909 946 834 952 984 861 816 795 809
00:41:21 23.2 9 8 9 9 9 9 9 8 8 11 8 8 407 380 270 292 304 239 256 228 203 188 161 146 883 1037 1023 895 882 824 923 968 803 843 789 795
00:46:21 23.2 11 8 9 8 8 9 8 12 8 11 8 8 413 364 266 272 269 220 240 222 192 169 170 149 865 992 978 868 889 790 882 894 784 779 780 797
00:51:21 23.3 9 9 8 13 9 8 9 10 8 8 12 9 383 355 266 247 274 227 222 213 189 151 162 140 821 933 964 864 849 749 871 875 819 782 759 741
00:56:21 23.3 9 9 8 10 9 8 9 9 9 8 10 9 388 369 269 272 266 204 222 195 182 166 137 138 823 918 952 835 800 726 835 846 787 765 716 746
01:01:21 23.3 9 10 9 9 9 8 9 9 9 8 9 8 379 349 235 228 273 221 208 184 164 158 145 133 755 884 879 821 788 687 850 902 779 731 727 746
01:06:21 23.3 8 10 9 10 9 9 9 11 9 8 9 10 376 340 223 238 263 208 194 195 176 152 140 121 745 851 855 760 804 697 809 834 754 722 710 715
01:11:21 23.3 9 8 9 8 8 10 8 9 10 9 8 9 346 339 238 216 242 211 190 175 162 138 131 119 739 838 843 765 739 708 783 791 739 713 720 732
01:16:21 23.3 9 9 8 9 9 9 9 9 11 10 9 9 345 345 189 218 247 199 189 165 149 127 135 133 722 820 799 754 730 650 770 792 680 708 680 703
01:21:21 23.3 8 8 10 10 9 10 10 8 9 9 8 10 345 319 203 214 250 195 188 169 148 148 127 127 720 794 794 746 716 646 707 788 716 679 663 678
01:26:21 23.3 9 9 9 9 9 10 10 9 9 10 10 10 347 326 199 212 244 189 189 159 159 128 136 121 693 799 779 720 697 619 753 762 698 650 645 695
01:31:21 23.3 9 9 9 9 9 8 9 9 9 11 9 9 326 309 188 216 225 187 180 157 140 117 125 113 659 775 738 709 702 607 726 737 699 642 630 670
01:36:21 23.3 9 9 9 8 9 9 9 9 9 8 8 8 347 295 170 197 224 180 168 157 148 114 109 100 668 722 733 678 664 580 666 700 633 655 598 647
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 97 635 683 699 652 651 577 679 729 647 604 585 607
01:46:21 23.3 9 8 9 10 8 8 10 8 9 10 10 11 302 293 160 181 220 170 167 144 130 120 113 109 660 713 701 622 640 574 657 650 621 607 579 606
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 98 113 106 603 676 662 610 624 564 641 673 607 582 550 597
01:56:21 23.3 9 8 8 10 9 8 9 10 9 10 10 10 283 257 147 161 194 159 139 138 123 108 115 108 609 656 660 610 594 540 651 625 590 564 550 584
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 94 118 102 598 652 626 627 586 524 618 630 575 561 551 598

Of all 4 measurements, the average was taken an is summarized in the chart below.

Figure 1: Average Luminescence Levels over time after 30 minutes incubation

As expected the control sample (UT) did not show any luminescence in the presence of the NanoLuc substrate.
eBAK1 coexpressed with eEFR showed a strong increase in luminescence. However, the highest levels were recorded in samples without the bacterial epitope elf18. This was unexpected as the epitope elf18 is the known elicitor to induce ligand-dependent interaction between the EFR receptor and its coreceptor BAK1 in A. thaliana. We later repeated the assay with the EFR ectodomain to get more data. Presence of the bacterial epitope elf18 did not lead to an increase in fluorescence levels again.

eBAK1 coexpressed with eCORE showed an increase in luminescence, although the effect was much smaller when compared with eEFR. The results again suggest, that addition of the bacterial elicitor csp22, which initiates interaciton between CORE and BAK1 does not increase the luminescence levels as samples without csp22 added showed greater luminescence than samples which were treated with this bacterial epitope.

These results suggest that our plasmids get expressed. It further has been shown that the NanoBit parts fused to the receptors are able to interact and reconstitute their functionality as a funcitonal NanoLuc protein which catalyzes the reaction of furimazine to furimamide, which gives a luminescent output. In our case, however, receptor-specific bacterial epitopes did not increase luminescence levels when the receptors were expressed in S. cerevisiae.

Second Assay

The assay was repeated in the a similar manner with the following differences:

  • We did not test again for samples which had been transformed with eCORE plasmids. So we only had samples for untransformed yeast cells and for cells transfected with the plasmids containting Part:BBa K3610038 and Part:BBa K3610043.
  • The optical density of all samples was adjusted to OD600 = 0.26.
  • Measurement started immediately after addition of the bacterial elicitor and the NanoGlo solution.
Figure 2: Average Luminescence Levels over time, measured directly after addition of bacterial elicitor and furimazine

Our observations of the second luminescence assay matched our expectations after the first one. Luminescence levels were indeed increased when the cells had previously been transformed with our constructs. It was again the case, however, that reconstitution of the NanoLuc protein was not driven by the ligand-dependent interaction of the plant receptors. It seemed, again, to be the case, that addition of bacterial elicitor did in fact decrease the measured luminescent output.

Third Assay

The assay was repeated again with the following differences (compared to the first assay):

  • We did not test again for samples which had been transformed with eCORE plasmids. So we only had samples for untransformed yeast cells and for cells transfected with the plasmids containting Part:BBa K3610038 and Part:BBa K3610043.
  • NanoGlo was added before the measurements were made, the bacterial elicitor was added right before measurement
  • Optical density was adjusted to OD600 = 0.5. We waited, however, for almost two hours, during which the cells were kept in TE buffer. During this time, a lot of the cells died, which decresaed the amount of whole living cells as TE supports cell lysis.
  • Only one bacterial epitope (elf18) was added.
Figure 3: Average Luminescence Levels over time, furimazine added 30 minutes before measurement and bacterial elicitor added immediately before.

A third time, we were able to observe that expression of our constructs increased luminescence intensity when the substrate furimazine was added. We, additionally, observed that the increase in luminescence was not initiated or significantly enhanced by presence of the bacterial epitope elf18.

Summary of Luminescence Assays

We conducted for assays with a plate reader of the type Synergy H1. Each time, luminescence was strongly increased when the samples had been transfected with plant PRRs fused to the NanoBits. This has especially been the case when the plant recpetors EFR and its coreceptor BAK1 were used. The increase in luminescence intensity did not seem to be driven by epitope.dependent interaciton of the two receptors. It is possible that, unlinke plants, S. cerevisiae cells lack any mechanisms to prevent unprovoked interaction between the target-receptor EFR and BAK1. Another possible explanation for the results could be that the receptors proteins are not processed the same way as they are in plant cells. For example, in plant cells, the receptor proteins get highly glycosylated. It is possible that this glycosylation influences the structure and the functionality of the receptor proteins.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 848
    Illegal PstI site found at 893
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 848
    Illegal PstI site found at 893
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1278
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 848
    Illegal PstI site found at 893
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 848
    Illegal PstI site found at 893
  • 1000
    COMPATIBLE WITH RFC[1000]


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