Difference between revisions of "Team:Wageningen UR/Results/Fluorescent"
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<b>Figure X:</b> Graph showing the relative fluorescence of the split fluorescent protein in comparison to their full length versions. | <b>Figure X:</b> Graph showing the relative fluorescence of the split fluorescent protein in comparison to their full length versions. | ||
Revision as of 19:00, 30 October 2017
Choosing the best reporter
The aim of this project is to analyze different reporter proteins to choose the one with the most suitable characteristics for our device. As Mantis's signal generation is based on bimolecular fluorescence complementation, the candidate reporter proteins were split and their halves fused to synthetic leucine zippers in order to promote their reassembly. The best reporter should fulfill a set of characteristics. First, the reporter protein has to show a bright signal, so our device can easily detect if the system has been activated. Second, the reassembly and maturation of the split reporter must be a fast as possible, to be able to detect a signal in a short timeframe. Last, our device will be used in tropical areas, where temperatures can be really high. Therefore, the reporter must also be able to produce a strong signal and mature fast at high temperatures.
Bimolecular fluorescence complementation or BiFC is a technique used to study and detect protein-protein interactions. It is based on the development of protein fragments which will reassemble when brought together by fusion proteins. This technique has substituted others such as Förster resonance energy transfer (FRET) or two-hybrid systems due to its flexibility, less background and direct analysis. There are several criteria that a split-protein system must accomplish in order to provide a successful tool [1]. Among these, the reassembled protein should present the desired activity with sufficient intensity to be detected and measured. Besides, the individual fragments should not present any signal and there should not be any affinity between them, providing a low signal/noise ratio. This way, the split protein will only reassemble when the two halves are brought together by the fused, interacting proteins (Figure X).
Among the different proteins that have been used in split protein systems, the fluorescent proteins have been the most widely used. However, not all fluorescent proteins are equally suitable for BiFC analysis. One characteristic that is essential when using split protein reassembly is a monomeric conformation, as any other oligomeric state will render a lack of signal when a monomeric subunit is reassembled. Furthermore, a multimeric conformation might lead to unwanted interaction among the proteins, precipitation and even toxicity for the cell [2]. The ideal fluorescent protein should have a high quantum yield, high photostability and a short maturation time [3].
Choosing candidate reporter proteins, their split sites and the interacting proteins
Since our signal is based on bimolecular complementation, we selected proteins that could be split in two parts and were able to regenerate a functional protein when coming together. Several fluorescent proteins had been previously split to be used as reporters in Bimolecular Fluorescence Complementation (BiFC) [4]. We chose five of them as our candidates: mRFP (Red fluorescent protein, BBa_K2387054), sfGFP (Green fluorescent protein, BBa_K2387047), eYFP (Yellow fluorescent protein, BBa_K2387003), mVenus (Yellow fluorescent protein, BBa_K2387045) and mCerulean (Cyan fluorescent protein, BBa_K2387052). We based our decision on their availability either in the iGEM Registry or in our laboratory. In addition to fluorescent proteins, we also thought on the possibility of using split chromoproteins in Mantis so the signal could be appreciated with the naked eye and without using any artificial source of light. Although there is a large collection of chromoproteins in the iGEM Registry, none of them are monomeric. After a literature research, we found three monomeric chromoproteins: Dathail [5] (Orange chromoprotein), Ultramarine [6] (Blue chromoprotein) and anm2CP [7] (Purple chromoprotein, BBa_K2387001).
In order to choose the split sites we carried out a literature research. However, to our knowledge, chromoproteins have never been split. Therefore, our approach on this case was to look at the structures of the chromoproteins or related proteins and to choose a split site situated in the loop between the eighth and the ninth β-strands. The structure of Dathail is available on PDB (5EXU). For Ultramarine, we looked at the structure of Rtms5 (2P4M), the multimeric chromoprotein from which it was evolved [6]. For anm2CP we looked at the structure of Killer Red (2WIQ), a phototoxic fluorescent protein derived from anm2CP [8]. The list with all the selected split sites can be found in Table A.
| Table A: Chosen split sites. | |||
|---|---|---|---|
| Protein | Split Site(s) | Position | Reference |
| anm2CP | 154-155
168-169 |
Loop between β7 & β8 strands
Loop between β8 & β9 strands |
2WIQ |
| Dathail | 165-166 | Loop between β8 & β9 strands | 5EXU |
| Ultramarine | 169-170 | Loop between β8 & β9 strands | 2P4M |
| mRFP | 154-155
168-169 |
Loop between β7 & β8 strands
Loop between β8 & β9 strands |
iGEM Registry (BBa_I715022 & BBa_I715023)
[9] Guido, J., et al. (2006) |
| eYFP | 154-155 | Loop between β7 & β8 strands | [10] Chang-Deng, H., et al.(2002) |
| mVenus | 155-156 | Loop between β7 & β8 strands | [11] Shyu, Y.J., et al. (2006) |
| sfGFP | 214-215 | Loop between β10 & β11 strands | [12] Zhou, Jun, et al. (2011) |
| mCerulean | 155-156
172-173 |
Loop between β7 & β8 strands
Loop between β8 & β9 strands |
iGEM Registry (BBa_K157006 & BBa_K157005)
[11] Shyu, Y.J., et al. (2006) |
One of the most common proteins chosen in literature to test bimolecular complementation are leucine zippers. These are simple protein domains widely spread in nature that interact among themselves. The leucine zippers found in nature interact in a parallel fashion, having both C-terminal ends at one side and the N-terminal ends at the other side. In order to use parallel leucine zippers for bimolecular complementation, both N-terminal ends or C-terminal of the split fragments ends should be proximal in the reassembled structure. Although this happens for some split sites situated in the same side of the protein were the natural ends are found, other split sites are found on the other side of the protein. This situation requires usage of antiparallel leucine zippers. Tho only antiparallel leucine zippers used in bimolecular complementations are in the fact the ones that were used in the first biomlecular complementation experiment with GFP. These leucine zippers are synthetic sequences and were used joining them through a Gly-Ser linker to the split fragments. We used these same leucine zippers for inducing the reassembly of our split reporters.
Constructs
The sequences of the full length proteins were PCR amplified using as templatesBiobricks from the iGEM Distribution Kit (BBa_E1010, BBa_K525406, BBa_I746916), Addgene Plasmid #27794mVenus C1 or gBlocksⓇ purchased from IDT. The fragments for the split proteins were amplified from the sequence of the full length protein. The fragments were fused to the respective leucine zippers through Golden Gate Assembly, introducing, in way that the two protein domains are fused by a glycine and a serine. The full length proteins, as well as both halves of the split proteins fused to the leucine zippers were cloned in pSB1C3 under the control of the araC/pBad promoter (BBa_I0500) and the strong RBS BBa_B0034. As example,a plasmid containing both fragments of sfGFP can be observed in Figure A.
These plasmids were transformed into E. coli DH5α. After single colonies were selected, the plasmids were extracted and sent for sequencing to verify the sequence. Once the sequence was corroborated, plasmids were transformed in E. coli BL21(DE3). This strain was the one used to perform the experiments.
Chromoproteins
A set of monomeric chromoproteins (anm2CP, Ultramarine, Dathail) and monomeric fluorescent proteins (mRFP, eYFP, mVenus, mCerulean, sfGFP) was successfully transformed and expressed in E. coli BL21(DE3). At the start of our project, we were considering the use of chromoproteins as a possible reporter that would allow to detect the signal with the naked eye. Inducing the expression of the reporters using arabinose allowed us to observe potential candidates (Figure 1).
Among the chromoproteins only anm2CP (BBa_K2387001) produced colored cells. Among the fluorescent proteins, mRFP (BBa_K2387054) produced bright pink cells and mCerulean (BBa_K2387052) produced bright yellow cells. The absorbance spectra of these proteins and their split versions were analyzed and Table 1 shows the absorbance at the maximum absorbance wavelength for each of them.
| Table 1: Values of Absorbance at the picks maximum values. | |||||
|---|---|---|---|---|---|
| Protein | Wavelength | Max. Absorbance (Full length) | Part number (Full length) | Max. Absorbance (Split) | Part number (Split) |
| anm2CP | 570 nm | 0.313±0.007 | BBa_K2387001 | -0.032±0.005 | - |
| mCerulean | 440 nm | 0.6±0.03 | BBa_K2387052 | 0.09±0.04 | BBa_K2387053 |
| mRFP | 590 nm | 0.59±0.05 | BBa_K2387054 | 0.06±0.02 | BBa_K2387055 |
The data on the table shows that the absorbance of the split proteins is really low compared to their full-length version. This correlates with the fact that colonies that expressed the split proteins didn’t show color detectable by the naked eye. For this reason we decided to develop our device to measure fluorescence and we continued characterizing our successfully split fluorescent proteins!
Testing Fluorescent Proteins
Once all the full length and split proteins were cloned in E. coli and their sequence confirmed, they were tested. This was checked by measuring their absorbance and fluorescence spectra. The absorbance and emission spectra for full mCerulean (BBa_K2387052) and split mCerulean (BBa_K2387053) can be observed in Figure B.
Comparison of the two emission spectra depicted in Figure B shows that the shape of the absorbance and emission spectra of mCerulean is not affected upon split. However, a proper recover of signal was not achieved for mRFP (BBa_I715022 & BBa_I715023) and mCerulean (BBa_K157006 & BBa_K157005) when they were split using the sites found in the iGEM Registry (155-156 for both). The spectra for the rest of fluorescent proteins can be found in their BioBrick Pages (Table X).
Although the Specific Visualization system requires the proteins to interact inside the cell in vivo, the Quorum Sensing system requires an extracellular signal. Therefore, we tested if the split fluorescent proteins could reassemble in vitro, after cells have lysed.
To test the in vitro reassembly, two plasmids were constructed, each of them containing one half of the split mVenus fused to the corresponding leucine zipper. The plasmids were transformed into E. coli DH5α. This way, two populations were obtained, each of them expressing one of the fragments. Before lysing the cells, the two populations were mixed in the same tube. E. coli DH5α without any plasmid was used as negative control, and E. coli DH5α expressing the full mVenus was used as positive control. The lysis procedure was enzymatic, using lysozyme, DNase and B-PER™ Bacterial Protein Extraction Reagent. Two variations of the protocol were tested: adding protease inhibitor, and not removing cell debris (not centrifuging). The lysate was measured to detect fluorescence emitted by mVenus (Figure C).
As can be observed, no fluorescence was detected arising from the sample with the two halves of the split mVenus, whereas fluorescence was detected in all variants of the protocol for the sample containing the full mVenus. This experiment proves that reassembly of split proteins in vitro does not happen (at least in the tested conditions).
Comparing Fluorescent Proteins
The first characterization we performed was a comparison between the performance of split fluorescent proteins versus the full length versions. For this test, we grew E. coli expressing split and full length fluorescent proteins in M9 minimal medium while measuring at the same time the evolution of fluorescence. The relative fluorescence of the split fluorescent proteins in relation to the full length proteins was calculated comparing the maximum values of fluorescence (Figure X).
As can be observed, split mVenus and split sfGFP excel in comparison to the other proteins, showing that the reassembly of the split portions of these proteins is more efficient than for other ones.
The next factor we took into account was the maturation rate of the fluorescent proteins. This rate can be calculated as the half-time obtained from the evolution of fluorescence over time, showed in Figure 2. The maturation rates can be found in Table 2.
| Table 2: Maturation rates determined as t1/2 (min). | |||||
|---|---|---|---|---|---|
| Protein | QY Full Protein | Part Number (Full length) | QY Split Protein | Part Number (Split) | |
| mRFP | 32±1 | BBa_K2387054 | 54±3 | BBa_K2387055 | |
| eYFP | 18±4 | BBa_K2387003 | 19±1 | BBa_K2387065 | |
| mVenus | 41±5 | BBa_K2387045 | 59±8 | BBa_K2387046 | |
| sfGFP | 20.7±0.7 | BBa_K2387047 | 25±7 | BBa_K2387048 | |
| Cerulean | 11.6±0.9 | BBa_K2387052 | 22±4 | BBa_K2387053 | |
According to the results, mVenus is the fastest one although the difference with mCerulean and sfGFP is not too big. Therefore, one of these three proteins would be a preliminary good candidate for our device.
As an additional parameter to choose the best reporter we calculated the Quantum Yields (QY) of the split fluorescent proteins, which relates the light absorbed and the emitted fluorescence. Therefore, the QY can be used as an estimate of the brightness of the fluorescent protein. The QY must be calculated in comparison to a reference using the same wavelength for excitation. The full proteins were used as standards for the split proteins, as their QY has already been calculated previously. The calculated QY can be found in Table 3.
| Table 3: Quantum Yields (QY) calculated for split proteins. | |||||
|---|---|---|---|---|---|
| Protein | QY Full Protein | Reference | Part Number (Full length) | QY Split Protein | Part Number (Split) |
| mRFP | 0.15 | [9] Guido, J, et al. (2006) | BBa_K2387054 | 0.06±0.04 | BBa_K2387055 |
| eYFP | 0.61 | [13] Nagai, T., et al. (2005) | BBa_K2387003 | 0.004±0.027 | BBa_K2387065 |
| mVenus | 0.57 | [13] Nagai, T., et al. (2005) | BBa_K2387045 | 0.61±0.06 | BBa_K2387046 |
| sfGFP | 0.65 | [14] Pédelacq, J., et al. (2004) | BBa_K2387047 | 1.3±0.2 | BBa_K2387048 |
| Cerulean | 0.62 | [15] Rizzo, M. A., et al. (2004) | BBa_K2387052 | 0.51±0.08 | BBa_K2387053 |
The results show that the split sfGFP is even better than the full sfGFP. This may be due to background noise in the absorbance of the full sfGFP, which would alter the results. However, although it is likely that splitting sfGFP does not increase the QY, the high value of QY for the split sfGFP show that this split protein can be considered as bright. Among the other proteins, mRFP and eYFP show much lower QY when compared to the full proteins. Both mVenus and mCerulean show QY values similar to that of the full proteins, which indicates that these two proteins are good candidates in terms of brightness.
As mVenus seemed to be the best fluorescent protein for Mantis when taking into consideration its brightness and its maturation time, we implemented it in the Specific Visualization system to reduce the signal detection system. Unfortunately, the system did not show the expected results (See the results of the experiment here).
The last characteristic we wanted our report to has was thermostability. Our split reporter should be able to mature and produce fluorescence at high temperatures, typical of tropical areas (25°C-35°C). We tested the evolution of fluorescence for 3 hours at different temperatures: 4°C, 10°C, 20°C (Room Temperature), 30°C, 45°C (possible temperature in tropical regions) and 60°C. The results are shown in Figure 3.
ns: No significance (P>0.05); *: P≤0.05; **: P≤0.01; ***: P≤0.001.
The graph shows that two split proteins generate the most fluorescence when incubated at 45°C: mRFP and sfGFP, both in their full-length and split versions However, for mCerulean the full protein and its split version showed different behaviours. While full-length mCerulean matures best at 45°C, its split version shows more fluorescence at 20°C. This difference may be caused by a lower structural stability of the split fragments of mCerulean at higher temperatures. As split sfGFP has a maturation time of Xmin (at 30°C), is bright and it is thermostable at 45°C, it is our perfect candidate. We could not implement it in the Specific Visualization system due to time constraints, but we think that the use of this reporter will improve Mantis.
Conclusions
We analyzed two types of reporters: chromoproteins and fluorescent proteins. The first ones had not been used before in bimolecular fluorescence complementation. It was observed that they are not proper reporters for use in bimolecular complementation signalling. Five fluorescent proteins were successfully split, being able to detect fluorescence when reassembled inside the cells (in vivo) The fluorescent proteins tested and the split fluorescent proteins were submitted to the Registry. Both mCerulean and mRFP have been improved due to the fact that the split proteins already present in the Registry are split in the wrong site. The reassembly in vitro was tested with negative results. The early experiments showed promising results for mVenus and it was implemented in the specific visualization system. Both sfGFP and mCerulean have been shown to be bright and have fast maturation. However, among these only sfGFP is able to mature efficiently at 45°C and therefore is the one we choose for our device.
References
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- Shaner, N.C., et al., "A guide to choosing fluorescent proteins." Nature Methods 2.12 (2005): 905–909.
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- Miller, Kristi .E., et al., "Bimolecular fluorescence complementation (BiFC) analysis: advances and recent applications for genome-wide interaction studies." Journal of Molecular Biology 427.11 (2015): 2039–2055.
- Langan, P.S., et al., "Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail." Journal of Molecular Biology 428.9 (2016): 1776–1789.
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- Bulina, Maria E., et al., "A genetically encoded photosensitizer." Nature Biotechnology 24.1 (2006): 95-99.
- Guido, Jach, et al. "An improved mRFP1 adds red to bimolecular fluorescence complementation." Nature Methods 13.8 (2006): 597-600.
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- Shyu, Y. John, et al. "Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions" BioTechniques 40.1 (2006): 61-66.
- Zhou, Jun, et al. "An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein" Acta Biochim Biophys Sin 43.3 (2011): 239-244.
- Nagai, Takeharu, et al. "A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications" Nature Biotechnology 20 (2002): 87-90.
- Pédelacq, Jean-Denis, et al. "Engineering and characterization of a superfolder green fluorescent protein" Nature Biotechnology 24 (2004): 79-88.
- Rizzo, Mark A., et al. "An improved cyan fluorescent protein variant useful for FRET" Nature Biotechnology 22 (2004): 445-449.
