Part:BBa_K2387054

mRFP controlled by inducible araC/pBAD promoter

mRFP is a red monomeric fluorescent protein. In this composite, its expression is regulated by the araC/pBad promoter and by a strong RBS. mRFP presents a maximum pick of excitation at 584 nm and a maximum pick of emission at 607 nm. mRFP was engineered from DsRed to grant it a monomeric conformation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1793
  Illegal AgeI site found at 1905
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Functional Parameters

In order to test the functionality of this protein under the araC/pBad promoter, the absorbance and the fluorescence spectra were measured after induction with 0.02% arabinose (Figure 1).

Figure 1: Absorbance and fluorescence spectra of mRFP, measured after induction with 0.02% arabinose.

This protein is part of a collection of split proteins developed for BiFC analysis and tested with the CpxR system (BBa_K2387032). These proteins were characterized through multiple experiments using as interacting proteins antiparallel synthetic leucine zippers. The proteins of this collection are the ones found in Table 1.

Table 1: Split Proteins.
Protein	Part Number (Full length)	Part Number (Split Protein)
mRFP	BBa_K2387054	BBa_K2387055
eYFP	BBa_K2387003	BBa_K2387065
mVenus	BBa_K2387045	BBa_K2387046
sfGFP	BBa_K2387047	BBa_K2387048
mCerulean	BBa_K2387052	BBa_K2387053

For comparing our split fluorescent proteins, the first factor we determined 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 2).

Figure 2: Graph showing the relative fluorescence of the split fluorescent protein in comparison to their full length versions.

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.

Another parameter we measured to choose the best reporter was 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 estimation for the brightness of the fluorescent proteins. 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 2.

Table 2: 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	Guido, J, et al. (2006)	BBa_K2387054	0.06±0.04	BBa_K2387055
eYFP	0.61	Nagai, T., et al. (2005)	BBa_K2387003	0.004±0.027	BBa_K2387065
mVenus	0.57	Nagai, T., et al. (2005)	BBa_K2387045	0.61±0.06	BBa_K2387046
sfGFP	0.65	Pédelacq, J., et al. (2004)	BBa_K2387047	1.3±0.2	BBa_K2387048
mCerulean	0.62	Rizzo, M. A., et al. (2004)	BBa_K2387052	0.51±0.08	BBa_K2387053

As observed in Table 2, mRFP and eYFP show much lower QY when compared to the full proteins. Both mVenus and mCerulean show QY values similar to those of the full proteins, which indicates that these two proteins are good candidates in terms of brightness. However, 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. Nevertheless, 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 bright.

The next factor we took into account was the maturation rate of the fluorescent proteins. This rate can be calculated as the half-time, which is the time at which half of the maximum fluorescence is reached. The maturation rates can be found in Table 3.

Table 3: Maturation rates determined as t1/2 (min).
Protein	Full Protein (min)	Part Number (Full length)	Split Protein (min)	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
mCerulean	11.6±0.9	BBa_K2387052	22±4	BBa_K2387053

According to the results, eYFP and mCerulean are the fastest ones although the difference with sfGFP is not too big. However, we have already observed that eYFP shows a weak fluorescence intensity, so the time it takes it to reach a detectable fluorescence will be longer than the one for sfGFP and mCerulean.

The last characteristic we wanted our report to has was thermostability. 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 incubation for three hours at different temperatures. A t-test was used to analyze differences between the values at 20°C (room temperatures) and the values at the other temperatures.

The graph shows that two of the 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.