Coding

Part:BBa_K3168002

Designed by: Eva Hanckmann, Harm van der Veer, Claire Michielsen   Group: iGEM19_TU_Eindhoven   (2019-09-12)
Revision as of 12:30, 27 October 2020 by Jtrott (Talk | contribs) (→‎Assay 1)

LargeBitNanoLuc

This basic part codes for the large bit of NanoLuc, which can be used as a split reporter. In combination with the small bit of NanoLuc and the addition of furimazine (the substrate), bright blue light is emitted (Figure 1). A flexible (GGS)5 linker is located in front of the large bit to enable the formation of fusion proteins. A strep-tag is also included at the end for protein purification.

T--TU Eindhoven--Split-NanoLuc.png

Figure 1. Split-NanoLuc principle.

Usage and Biology

Split reporters have been used a lot to detect protein-protein interactions and for screening purposes (Dixon, 2015). NanoLuc is a small, structurally robust and very bright luciferase. Many methods have been developed using the split variant of NanoLuc, such as the HiBiT technology and NanoLuc ternary technology (Ohmuro-Matsuyama, 2019).


2020 Improvement UZurich

For our 2020 iGEM project, we from the University of Zürich designed a novel approach for a biosensing system relying on plant pattern-recognition receptors (PRRs). We managed to engineer parts of the system in the lab and conduct initial experiments to characterize our system. After many discussions with experts, we decided to use the split-NanoLuc system to generate a luminescent output. Since our system was designed for our chassis S. cerevisiae we decided to use a codon optimized sequence for the output, as we wanted to facilitate expression in our chassis. For this purpose, we codon optimized this part and also Part:BBa K3168003, which encodes for the SmallBit, the second half of the functional NanoLuc luciferase. The registry numbers for the codon optimized parts are : BBa_K3610014 for LargeBit and BBa_K3610014 for SmallBit. As we were using these parts in our project, we decided to test our improved parts and compare them with the original sequences.

Approach

Our goal was it to test whether expression of the codon optimized parts in S. cerevisiae would enhance luminescence intensity of a sample when the NanoLuc substrate furimazine is added.

We tackled this question by using rapamycin and its binding ligands, FKBP and FRB (Part:BBa_K3610022, Part:BBa_K3610023).

FKBP

he FK506 binding protein, or FKBP, is a protein belonging to the immunophilin family and has proven to be a useful tool in biological research. Functionally, the protein is a peptidyl-prolyl cis-trans isomerase (PPI).
Responsible for its prominent role in research is the ability of FKBP to bind to rapamycin, an antifungal antibiotic macrolide. What makes this interaction interesting is the fact that rapamycin binds to FKBP and the FKBP–rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) simultaneously, inducing a dimerization of these two components. These properties of FKBP and FRB have been exploited for conditional dimerization of proteins of interest, which offers many possibilities to artificially manipulate cellular processes, by fusing them to FKBP and FRB.

FRB

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase, which is involved in the control of mRNA translation initiation. The FKBP12-rapamycin binding domain (FRB) is located upstream of the catalytic domain and is essential for the kinase activity of the mTOR protein. Without the FRB domain the protein loses its functionality. As already established the interesting property of FRB is its interaction with the antifungal antibiotic macrolide rapamycin. Rapamycin binds to FRB and the FK506 binding protein (FKBP) simultaneously, inducing dimerization of the two components. This rapamycin-induced dimerization has been exploited for dimerization of proteins of interest by fusing them to FKBP and FRB.

Rapamycind binding ligands and NanoBits

The previously described properties come in handy when we need to induce protein-protein interaction. We decided to test the improved construct in a dimerization assay with rapamycin.
For this purpose, we designed the following constructs:

The split protein was fused to either FKBP12 or FRB via a 15 amino acid(AA)-long linker consisting of the small AAs Glycin and Serine. Additionally, the two LargeBit constructs contain a hemagglutinin tag at between the receptor protein-coding region and the 15AA-linker, which is used for (co-)immunoprecipitation and Western Blotting. The SmallBit constructs have a FLAG tag at the same position for the same purpose.

For testing our newly created part Part:BBa_K3610054, we co-expressed LBit:FKBP and SBit:FRB in <S. cerevisiae (AP4)</i> and did the same for LBit:FKBP_yeast and SBit:FRB. When both parts are expressed, addition of rapamycin will make the ligands FKBP12 and FRB bind to it, which will initiate the interaction between the LargeBit and the SmallBit. Since SmallBit is very short and has hardly any codons that are rare in S. cerevisiae, we only tested the codon optimized LargeBit. Should codon optimization actually facilitate expression in yeast, we would expect the luminescence intensity to be higher, when rapamycin and the substrate furimazine are added.

Preparation of the constructs

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.

We expressed the S. cerevisiae cells with two different plasmids, each containing one construct. One plasmid with a -TRP gene, enabling the yeast strain to produce tryptophan, the other contains a gene for the kanamycin acetyltransferase. We therefore needed a synthetic dropout medium (-TRP) supplemented by Geneticin.

Procedures

After successful transformation, we examined the cells with a plate reader of the type Synergy H1. Cells were inoculated in liquid medium for several hours at 30°C and adjusted to the same OD600. This step was necessary to prevent differences in measured values due to higher cell density in one sample.

Afterwards, the 96 well plate was prepared with 50μl samples containing LBit and samples containing LBit_yeast. We divided the samples into groups, those to which no rapamycin was added and those that received additional rapamycin. The substrate furimazine was added to each well in form of 50μl NanoGlo solution (50:1 buffer to substrate). The rapamycin was added immediately after addition of furimazine. 10μM rapamycin was added, giving a final concentration of 10 μM.

Further Details:

  • Runtime 2:00:00 (HH:MM:SS)
  • Interval 0:06:00, 21 Reads
  • Shake
    • Linear:
      • Continuous
      • Frequency: 493 cpm (4 mm)
    • Read
      • Luminescence Endpoint
      • Full Plate
      • Integration Time: 0:03.00 (MM:SS.ss)
      • Filter Set 1
        • Emission: Full light
        • Optics: Top, Gain: 135
      • Read Speed: Normal, Delay: 100 msec
      • Extended Dynamic Range
      • Read Height: 1 mm

Results

Assay 1

In the first measurement series, all samples were adjusted to OD600 = 0.26. The following samples were prepared:

  • 6X LBit_yeast without rapamycin
  • 6X LBit yeast with rapamycin
  • 6X LBit registry without rapamycin
  • 6X LBit registry with rapamycin

(LBit registry referres to this part)
Measurements with a luminometer at 480 nm over two hours gave the following results (T° Lum for each measurement between 23.9 and 24.1).

Time Yeast Yeast Yeast Yeast Yeast Yeast Yeast + rapamycin Yeast + rapamycin yeast + rapamycin yeast + rapamycin yeast + rapamycin yeast + rapamycin registry registry registry registry registry registry registry + rapamycin registry + rapamycin registry + rapamycin registry + rapamycin
00:00:45 2430 2106 2092 1994 1895 1710 49039 32120 13199 20462 22483 24117 1042 970 954 1018 877 903 20088 20125 29619 24759
00:06:45 3271 2816 2911 2204 2315 2026 46768 30463 13767 19967 21892 21831 1310 1244 1272 1294 1217 1212 20065 19926 26624 21277
00:12:45 3214 2820 2889 2178 2244 2056 43677 28730 13313 18920 20964 20778 1550 1545 1580 1569 1493 1479 19268 19546 25222 19791
00:18:45 3023 2650 2664 2287 2305 2100 41396 27459 12911 18189 20333 20162 1812 1921 1997 1826 1806 1792 18789 19224 24308 19327
00:24:45 2763 2467 2459 2330 2314 2070 39354 26259 12778 17623 19618 19525 2185 2420 2639 2278 2202 2252 18502 19078 23598 18899
00:30:45 2552 2270 2301 2248 2260 2021 38112 25162 12574 17283 19360 18904 2629 3095 3486 2799 2771 2797 17938 18721 22991 18565
00:36:45 2365 2085 2082 2094 2104 1941 37227 24339 12385 16842 18745 18397 3188 3925 4386 3451 3456 3517 17790 18598 22435 18363
00:42:45 2234 1939 1928 1979 2006 1849 36265 23546 12096 16723 18341 17976 3827 4780 5369 4094 4136 4256 17356 18112 21884 18116
00:48:45 2090 1850 1851 1857 1884 1751 35178 22867 11895 16327 17988 17540 4400 5586 6271 4822 4857 4929 16951 17857 21400 17986
00:54:45 1923 1736 1720 1747 1801 1655 34509 22317 11848 16050 17505 17175 4890 6314 7066 5422 5473 5622 16600 17608 21021 17674
01:00:45 1812 1652 1609 1676 1705 1586 33544 21861 11442 15757 17115 16677 5431 7007 7811 6030 6111 6203 16252 17150 20335 17502
01:06:45 1756 1549 1505 1603 1626 1484 32964 21117 11353 15354 16719 16285 5945 7511 8433 6639 6739 6801 15953 16864 19968 17190
01:12:45 1670 1503 1495 1515 1551 1468 32209 20543 11228 14970 16261 15748 6463 8079 9036 7137 7257 7326 15692 16609 19538 16976
01:18:45 1592 1438 1430 1468 1485 1383 31574 20053 10939 14737 15890 15547 6883 8649 9555 7528 7735 7883 15338 16182 18899 16729
01:24:45 1526 1353 1366 1409 1441 1347 31014 19482 10631 14317 15546 15120 7310 9101 9996 8100 8217 8390 14870 15861 18607 16267
01:30:45 1444 1309 1321 1357 1372 1297 30195 18987 10583 14070 15144 14679 7581 9525 10558 8470 8582 8792 14594 15499 18019 16121
01:36:45 1420 1271 1277 1281 1305 1241 29662 18598 10246 13906 14769 14456 7899 9965 10959 8914 8873 9234 14334 15107 17597 15632
01:42:45 1360 1266 1249 1292 1278 1217 28843 18010 10022 13499 14429 14021 8216 10295 11263 9165 9292 9450 13909 14814 17252 15296
01:48:45 1284 1183 1183 1181 1241 1197 28291 17679 9855 13161 14198 13638 8449 10496 11649 9449 9695 9743 13500 14327 16701 15007
01:54:45 1266 1178 1139 1189 1238 1105 27502 17233 9583 12973 13746 13337 8616 10862 11888 9623 9843 10037 13216 14020 16099 14695
02:00:45 1218 1110 1131 1123 1182 1151 26890 16840 9330 12579 13572 12858 8885 10994 12129 9834 10045 10175 12851 13614 15693 14332

The measurement values are summarized in the graph below:

Figure 1: Average Luminescence levels over time

As expected, the control samples (no rapamycin added) gave hardly any detectable luminescent output. When rapamycin was added to the wells, increased luminescence intensity could be detected. This initial assay showed increased luminescence in samples transformed with LBit:FKBP_yeast (codon optimized). The variance, however, is too big to make any assumptions after this initial measurement (regarding increased luminescence when compared to the original sequence).

Assay 2

Because the first data from the first measurement was not very conclusive, the same type of measurement was repeated. With the following differences:

  • OD600 = 0.5 for all samples
  • longer waiting period between taking cells out of the incubator and measurement (2 hours)
  • After addition of NanoGlo solution, the samples were incubated at RT for 30 minutes.

The following samples were measured:

  • 3X yeast without rapamycin
  • 9X yeast with rapamycin
  • 3X registry without rapamycin
  • 3X registry with rapamycin
Figure 2: Average Luminescence levels over time. Second measurement.

Analysis

The patterns observed in the second experiment were similar to the ones observed in the first assay. Expression of the constructs was successful and rapamycin was able to drive the interaction between the LargeBit and the SmallBit part, which is made visible by the luminescent output after addition of the NanoLuc substrate furimazine.
As had already been previously observed, samples which expressed the codon optimized version of this part, showed higher average fluorescence intensity at all measurement points.
When comparing Yeast_Rapamycin and Registry_Rapamycin the overall average luminescence intensity was significantly different (W = 6669.5, p-value < 2.2e-16, Wilcoxon Ranked Signed Rank Test was conducted as the measurements were made with cells coming from the same plate).

Conclusion

We conducted initial tests to characterize the parts Part:BBa K3168002 and Part:BBa_K3610014. When SmallBit and LargeBit are fused to the two rapamycin binding ligands FRB and FKBP12 respectively and expressed in S. cerevisiae (AP4), addition of rapamycin is able to drive the interaction between the SmallBit and the LargeBit, leading to the reconstitution of their functionality as a luciferase. Addition of rapamycin and the substrate furimazine will lead to a luminescent output.
Additionally, initial measurements with a luminometer suggested a stronger overall luminescence intensity, when the codon optimized sequence of LargeBit was used.

References

Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., ... & Wood, M. G. (2015). NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS chemical biology, 11(2), 400-408.

Ohmuro-Matsuyama, Y., & Ueda, H. (2019). Protein-Protein Interaction Assays Using Split-NanoLuc. In Bioluminescence. IntechOpen.

Kolos, Jürgen M.; Voll, Andreas M.; Bauder, Michael; Hausch, Felix (2018): FKBP Ligands—Where We Are and Where to Go? In: Frontiers in Pharmacology 9, S. 1425. DOI: 10.3389/fphar.2018.01425.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 526
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None