Composite

Part:BBa_K3610043

Designed by: Jonas Sebastian Trottmann   Group: iGEM20_UZurich   (2020-10-05)
Revision as of 23:52, 22 October 2020 by Jtrott (Talk | contribs) (→‎Luminescence assay)


EFR ectodomain / SmallBit for S. cerevisiae

This part includes the ectodomain of the plant pattern recognition receptor EFR 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

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 firts 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.

Usage with NanoLuc

In this case, the C-terminal domain of EFR, 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 EFR 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 EFR nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, elf18-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 fluorescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR 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 elf18 epitope in water samples as the elf18 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 (eEFR) 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.

we tested whether the split-NanoLuc proteins would be able to interact, which would reconstitute its functionality. We further were interested in the interaction between the EFR and BAK1 ectoomain. EFR and BAK1 naturally interact upon binding of the epitope elf18. Should both receptors be expressed properly in the cell membrane of S. cerevisiae, the ligand will be able to bind to the EFR ectodomain. If this is sufficient to drive dimerization of the two receptor in S. cerevisiae, then the NanoLuc parts will be more likely to interact as well, which would lead to a higher amount of functional luciferase proteins in the cell, 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 TÝ Lum UT UT UT UT UT + csp22 UT + csp22 UT + csp22 UT + csp22 UT + elf18 Ut + elf18 UT + elf18 UT + 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 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 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 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 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 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 8 997 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 9 964 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 8 906 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 8 883 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 8 865 992 978 868 889 790 882 894 784 779 780 797
00:51:21 23.3 9 9 8 13 9 8 9 10 8 8 12 9 821 933 964 864 849 749 871 875 819 782 759 741
00:56:21 23.3 9 9 8 10 9 8 9 9 9 8 10 9 823 918 952 835 800 726 835 846 787 765 716 746
01:01:21 23.3 9 10 9 9 9 8 9 9 9 8 9 8 755 884 879 821 788 687 850 902 779 731 727 746
01:06:21 23.3 8 10 9 10 9 9 9 11 9 8 9 10 745 851 855 760 804 697 809 834 754 722 710 715
01:11:21 23.3 9 8 9 8 8 10 8 9 10 9 8 9 739 838 843 765 739 708 783 791 739 713 720 732
01:16:21 23.3 9 9 8 9 9 9 9 9 11 10 9 9 722 820 799 754 730 650 770 792 680 708 680 703
01:21:21 23.3 8 8 10 10 9 10 10 8 9 9 8 10 720 794 794 746 716 646 707 788 716 679 663 678
01:26:21 23.3 9 9 9 9 9 10 10 9 9 10 10 10 693 799 779 720 697 619 753 762 698 650 645 695
01:31:21 23.3 9 9 9 9 9 8 9 9 9 11 9 9 659 775 738 709 702 607 726 737 699 642 630 670
01:36:21 23.3 9 9 9 8 9 9 9 9 9 8 8 8 668 722 733 678 664 580 666 700 633 655 598 647
01:41:21 23.3 9 10 10 9 10 9 8 9 9 9 10 10 635 683 699 652 651 577 679 729 647 604 585 607
01:46:21 23.3 9 8 9 10 8 8 10 8 9 10 10 11 660 713 701 622 640 574 657 650 621 607 579 606
01:51:21 23.3 11 10 9 9 9 9 9 8 9 10 8 9 603 676 662 610 624 564 641 673 607 582 550 597
01:56:21 23.3 9 8 8 10 9 8 9 10 9 10 10 10 609 656 660 610 594 540 651 625 590 564 550 584
02:01:21 23.3 8 9 9 9 9 9 9 10 9 11 9 9 598 652 626 627 586 524 618 630 575 561 551 598

The average of each type of measurement is displayed in the figure below.

File 1: Average luminescence over time

As expected, the untransfected samples were not giving a luminescent output. Cells containing the plasmids with the two ectodomains displayed a strong increase in luminescence. However, the highest levels were measured when no bacterial epitope was added.

The results suggest that the plasmids had been taken up and get expressed in S. cerevisiae. It has also been shown that the split-proteins, which were fused to the receptors were able to interact and regain functionality as an enzyme. It seems, however, that this reconstitution is not driven by ligand-dependent interaction of the receptors. It is possible that the receptors are able to interact but lack any regulatory features which are otherwise present in plants, such as the BIR receptors. Another reason for these results could be the lack of glycosylation. In plants the receptors get heavily glycosylated. It is at this point unclear how exactly the receptors are glycosylated in S. cerevisiae and how this influences the functionality of the receptors.


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]


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