Team:Wageningen UR/Results/Fluorescent

Choosing the best reporter

The aim of this project is to analyze different reporter proteins to choose the best. As our project signal is based on bimolecular complementation, the reporter proteins will be split and analyzed under reassembly directed through synthetic leucine zippers. The best reporter will have to fulfill a set of characteristics:

  • First, the reporter protein has to show a bright signal, so the device can detect easily 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.

Choosing proteins and split sites

Our signal is based on bimolecular complementation, so we need to test proteins that can be split in two parts that when coming together will reassemble into a functional protein. Several fluorescent proteins had been previously split to be used as reporters in BiFC. We chose 4 to test them as possible candidates: mRFP (Red fluorescent protein), sfGFP (Green fluorescent protein), eYFP (Yellow fluorescent protein), mVenus (Yellow fluorescent protein) and mCerulean (Cyan fluorescent protein). We chose this proteins due to their availability in the iGEM Resgistry and in our laboratory. 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 (Orange chromoprotein), Ultramarine (Blue chromoprotein) and anm2CP (Purple chromoprotein).

In order to choose the split sites we based our decision on a literature research. This research revealed that, although some fragments of split proteins can be found in the iGEM Resgistry, not all of them are split at the correct site. Choosing the split site of the chromoproteins was harder, as they had never been split before. Protein structures of the chromoproteins or related proteins were used to choose a split site situated in the loop between the 8th and the 9th beta-strands. The structure of Dathail is available on PDB. The structure of Ultramarine was considered to be similar to that of Rtms5, the multimeric chromoprotein which it was evolved from. The structure of anm2CP was considered to be similar to that of Killer Red, a phototoxic fluorescent protein derived from anm2CP. The list of chosen split sites can be found in Table A.

Table A: Chosen split sites.
Protein Split Site Reference
anm2CP 168
Dathail 165
Ultramarine 169
mRFP 168
eYFP 154
mVenus 155
sfGFP 214
mCerulean 172

Additionally, anm2CP was also split in the residue 154, situated in the loop between the 7th and the 8th beta strands.

Constructs

The sequences of the full length proteins were PCR amplified from the source (Plasmid from iGEM Distribution Kit, Addgene Plasmid or gBlock synthetic DNA, see Part Pages). The fragments for the split proteins were amplified from the sequence of the full protein. The fragments were fused to the respective leucine zippers. The full proteins, as well as both fragments for the split proteins were cloned in pSB1C3 under the control of the araC/pBad promoter and a strong RBS. An example of a plasmid containing both fragments of mVenus 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 known to be the correct one, the plasmids were transformed in E. coli BL21 DE3. This strain was the one used to perform the experiments.

Functional test for split proteins

Once all the full and split proteins were cloned in E. coli and their sequence confirmed, they were tested to check their activity. Whereas none of the chromoproteins was successfully split, all the fluorescence proteins were. This was checked by measuring the absorbance and fluorescence spectra. The spectra for full mCerulean and split mCerulean can be observed in Figure B.

From these spectra can be observed that when a protein is split, the shape of the absorbance and fluorescence spectra remains the same, which is an indication of proper splitting. The spectra for the rest of fluorescent proteins can be found in their BioBrick Pages.

Reassembly of split proteins in vitro

Although the CpxR-CpxR visualization system requires the proteins to interact inside the cellin vivo, the Quorum Sensing system requires the a extracellular signal. 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 to the protocol were tested: adding protease inhibitor, and not removing cell debris (not centrifuging). Not adding DNase was tried, by a muccus was formed that hampered the analysis of the sample. 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).

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).

Figure 1: E. coli BL21 DE3 expressing chromoproteins and fluorescent proteins under induction with 0.2% arabinose.

Among the chromoproteins only anm2CP produced colored cells. Among the fluorescent proteins, mRFP produced bright pink cells and mCerulean produced bright yellow cells. The absorbance spectra of these proteins and their split versions were analyzed and in an attempt to compare quantitatively the color produced, the absorbance at the maximum pick was calculated and recorded (Table 1).

Table 1: Values of Absorbance at the picks maximum values.
Protein Wavelength Max. Absorbance Max. Absorbance (Split)
anm2CP
mCerulean 440 nm 0.1002±0.0008 0.0001±0.0001
mRFP 560 nm 0.053±0.001 0.0017±0.0003

It is important to consider that this values only help us to compare between the same protein in its full and split version, because the color of the protein is caused by the full range of absorbance. For example, mRFP presents two picks and therefore it is expected that the intensity of the larger pick will still be lower that the intensity of the pick from mCerulean, which only shows one pick. Thus, with this data we cannot claim that mCerulean shows a more intense color than mRFP. The data on the table shows that the absorbance of the split proteins is lower than the absorbance of the full proteins. This correlates with the fact that colonies that expressed the split proteins showed no color. Between this fact and the development of our measurement device, we decided to continue analyzing only fluorescent proteins.

The first 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 (t1/2)
mRFP
Figure 2: Evolution of fluorescence after cells stop producing protein due to addition of chloramphenicol/geneticin in high concentrations. The increase in fluorescence is only be due to the maturation of the full and split fluorescent proteins.

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 paramter 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 QY Split Protein
mRFP 0.25 0.1±0.06
eYFP 0.61 0.004±0.027
Venus 0.57 0.61±0.06
sfGFP 0.65 1.3±0.2
Cerulean 0.62 0.51±0.08

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 brigth. Among the other proteins, mRFP and eYFP show lower QY than the one of the full proteins. Both mVenus and mCerulean show a QY similar to that of the full proteins, which indicates that these two proteins are good candidates in terms of brightness. As we had good results in both experiments with mVenus, we implemented mVenus to the CpxR-CpxR BiFC system in order to try to observe a decrease in the time necessary to observe the signal. Unfortunately, the system did not work properly with Venus as a reporter due to high noise.

The last characteristics we wanted our report to have was thermostability. Our split reporter should be able to mature and produce fluorescence in high temperatures, typical of tropical areas. 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.

Figure 3: Differences of fluorescence after three hours at different temperatures. A statistical analysis using t-test was used to identify significant differences between the values at 20°C (room temperatures) and the values at other temperatures. ns: No significance(P>0.05); *: P≤0.05; **: P≤0.01; ***: P≤0.001.

The graphic show that only two split proteins generate most fluorescence at 45°C: mRFP and sfGFP. mCerulean is a curious case, as the full protein matures best at 45°C but the split protein as a maximum temperature of maturation at 20°C. This difference may be caused by the lower structural stability of the split fragments of mCerulean at higher temperatures. This lower structural stability does not take place in split mRFP and sfGFP due to the different sizes and sequence of the fragments between the fluorescent proteins. As split sfGFP has been proven to work at 45°C, as well as being bright and fast, it is our perfect candidate. We could not implement it in the CpxR-CpxR BiFC system due to time constrains, but we are positive that using this reporter will improve our device

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References

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  2. Sullivan, Lauren, et al. "Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device." PLoS neglected tropical diseases 7.2 (2013): e2087.
  3. Overath, P., et al. "Invariant surface proteins in bloodstream forms of Trypanosoma brucei." Parasitology Today 10.2 (1994): 53-58.