mRFP1-derived, altered to be a BioBrick by removing a PstI site and adding BioBrick ends. [mRFP1 was itself a derived from DsRed (via 33 mutations!)]
mRFP1-derived, altered to be a BioBrick by removing a PstI site and adding BioBrick ends. [mRFP1 was itself a derived from DsRed (via 33 mutations!)]
−
mCherry is one of several "second-generation" monomeric fluorescent proteins developed in Roger Tsien's laboratory at UCSD (''cf., Nature Biotechnology'' '''22''', 1567 - 1572 (2004).
+
mCherry is one of several "second-generation" monomeric fluorescent proteins developed in Roger Tsien's laboratory at UCSD (''cf., Nature Biotechnology'' '''22''', 1567 - 1572 (2004). PMID 15558047
+
−
<!-- Add more about the biology of this part here--see above (SCM 12/1/06)
===Usage and Biology===
===Usage and Biology===
+
Some strange experimental results that have been seen could be explained by an internal RBS + start. The 10th amino acid is a Met which is preceded by AGGAGGA(NNNN). This is almost a perfect consensus RBS so it seems quite likely that translation can begin 10 amino acids in. Note that mCherry was designed by fusing the N and C terminal regions of EGFP on to a mRFP variant (to increase tolerance to protein fusions). Thus, removing the first several amino acids is not expected to have much effect on fluorescence. If this is truly a strong internal RBS, then the identity of any attached RBS may have little effect. Also, one should be careful when making protein fusions. --[[User:Austin|Austin]]
−
<!-- -->
+
The copy as provided in the 2010 distribution is incorrect - it contains ~500 bp of something that is not mCherry between the VF2 and VR sites. You can get a functioning copy via PCR out of [[Part:BBa_J06702]].
This part has been improved to be the mCHERRY UNIT including a degradation tag named DBOX, a fluorescent gene mCherry and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. The whole part can be cut off by VIKA.
+
For more information of the improved part, please go to the page of [[Part:BBa_K1926013]].
+
+
===Improvement by USP_UNIFESP-Brazil===
+
The reporter mCherry has been codon-optimized for Chlamydomonas reinhardtii and it was successfully tested as a reporter in this chassis. We have made several measurements to show its fluorescence in Chlamydomonas reinhardtii as well its properties (Fluorescence excitation/emission spectrum). For more information of the improved part, please go to the page of [[Part:BBa_K2136016]].
+
+
===Improvement by Evry_Paris-Saclay 2017===
+
The reporter mCherry has been codon-optimized for Escherichia coli K12 ([[Part:BBa_K2448004]]) and it was successfully tested as a reporter in this chassis. We have used this part to build the Universal Biosensing Chassis ([[Part:BBa_K2448023|BBa_K2448023]] and [[Part:BBa_K2448024|BBa_K2448024]]) and test our Psicose Biosensors ([[Part:BBa_K2448025|BBa_K2448025]], [[Part:BBa_K2448026|BBa_K2448026]], [[Part:BBa_K2448027|BBa_K2448027]], [[Part:BBa_K2448028|BBa_K2448028]], [[Part:BBa_K2448029|BBa_K2448029]], [[Part:BBa_K2448030|BBa_K2448030]] and [[Part:BBa_K2448031|BBa_K2448031]]) as well as our Fructose Biosensor ([[Part:BBa_K2448032|BBa_K2448032]]).
+
For more information on the improved part, please go to the page of [[Part:BBa_K2448004]].
+
+
===Improvement by University of Minnesota 2017===
+
The internal RBS-like sequence in this mCherry reporter was removed, and the overall sequence was codon-optimized for E. coli K12. This new part [[Part:BBa_K2375001|BBa_K2375001]] should be more suitable for creating fusion proteins.
+
+
===Improvement by TAU_Israel 2021===
+
The codon sequence has been optimized for expression in Bacillus subtilis against E. coli using the Communique tool using several methods. For more information, refer to part pages [[Part:BBa_K3871001|BBa_K3871001]], [[Part:BBa_K3871002|BBa_K3871002]], [[Part:BBa_K3871003|BBa_K3871003]], [[Part:BBa_K3871004|BBa_K3871004]],[[Part:BBa_K3871005|BBa_K3871005]] and the 2021 TAU_Israel wiki, available [https://2021.igem.org/Team:TAU_Israel here.]
+
+
=== Improvement by UCopenhagen 2022 ===
+
+
We improved this mCherry protein by adding SnoopCatcher (part <partinfo>BBa_K4247009</partinfo>), so that it can now spontaneously form an irreversible isopeptide bond with any protein that contains the SnoopTag (part <partinfo>BBa_K4247008</partinfo>). By adding a functional SnoopCatcher to mCherry and validating the SnoopTag-Catcher system, we have successfully improved the part BBa_J06504. Thus, mCherry with the SnoopCatcher can bind to essentially any protein with a SnoopTag. Since mCherry is a widely used protein, we believe our improvement opens up many new uses of the protein among its repertoire of diverse biotechnological applications. For more information about our improved part, go to <partinfo>BBa_K4247025</partinfo>.
+
+
==Contribution: TU_Eindhoven 2019==
+
In this contribution, we futher characterized mCherry (BBa_J06504). mCherry was cloned into a pET28a(+) vector (containing an N-terminal 6xHis-tag) and subsequently expressed in BL21 (DE3) E. coli and purified using immobilized metal affinity chromatography (IMAC). Different concentrations of mCherry were made and an increase in color intensity was visible (Figure 1).
''Figure 1. Purified mCherry in different concentrations, showing a clear increase in color intensity.''
+
+
Consequently, the purified protein was analyzed on an SDS-PAGE (Figure 2) which shows a clear blob above 25 kDa, corresponding with 6xHis-tagged mCherry’s molecular weight of 29.3 kDa. The blob in between 10 kDa and 15 kDa is a truncated version of mCherry as described by Tripathi [1]. There are other bands visible so the purity is not optimal. However, the contrast between the huge blobs and the other bands is very big.
+
+
[[File:T--TU_Eindhoven--SDSmCherry.png|80px|]]
+
+
''Figure 2. SDS-PAGE of mCherry after purification.''
+
+
Following protein purification, the protein functionality was analyzed. Firstly, the excitation and emission of mCherry were measured within its absorption and emission range (Figure 3). This shows a clear excitation maximum at 587 nm and an emission maximum at 610 nm.
+
Secondly, the photostability of mCherry was measured following UV radiation up until thirty minutes to advance usage of mCherry in experiments (Figure 4). Exposure to UV light shows a decrease in fluorescence intensity. After 30 min, the intensity is almost halved compared to no UV exposure.
''Figure 3. Excitation and emission spectrum of mCherry.<font color="white">AAAAAAAAAA</font>Figure 4. Excitation spectrum of mCherry after different periods of UV radiation.''
+
+
==HUBU-WUHAN 2019-Characterization==
+
We characterized the bleaching effect in <i>zymomonas mobilis</i> using fluorescent microscopy Leica DMi8. We obtained image information through fluorescence microscope and analyzed it with the Rleative Flourescense Calculator in LAS X.
+
[[File:T--HUBU WUHAN--mCherry.gif|600px|thumb|center| GIF.1 Real-time imaging of <i>zymomonas mobilis</i>.]]
+
It is expressed in pEZ-15A plasmid with a PlacUV5 promoter. LAS Z was used to analyze the images and calculate the rate of photobleaching. We obtained a half-life period as t1/2=40.07s
+
[[File:T--HUBU WUHAN--curve of halflife.png|600px|thumb|center| Fig.1 Curve fit of relative intensity.]]
+
+
==Characterization: SMMU-China 2019==
+
In our project this year, we aim to develop a biomedical tattoo that could detect disease related molecules and output signals through accumulation of fluorescent proteins. We have also developed a software to analyze the intensity of the fluorescence so that this signal could reflect the disease conditions. To test whether this software could accurately measure the fluorescence and whether florescent proteins are sensitive enough to meet our conception, we carried out the following experiments:
+
First, we cloned this part (BBa_J06504) into eukaryotic expression vector pcDNA3.1 and transfected it into HEK293T cells through Lipofectamine 2000 transfection reagent. Twenty-four and 48 hours after transfection, bright red light could be observed under florescence microscopy (Figure. 1)
+
[[File:T--SMMU-China--mCherry 1.png|600px|thumb|center| Figure. 1 mCherry expression at 24 and 48 hours after transfection.]]
+
+
The transfected and untransfected HEK293T cells were digested at 24h by trypsin and centrifugated in microtubes. The cells in microtubes were used to mimic the biomedical tattoo and were further examined. Whereas the cells did not show apparent difference with negative control under white light (Figure. 2a), they emitted red light when exposed to exciting light from a mercury lamp (Figure. 2b). This result suggested that fluorescent proteins were sensitive reporters and could be distinguished even by naked eyes under exciting light.
+
[[File:T--SMMU-China--mCherry 2.png|850px|thumb|center| Figure. 2 Tattoo cells under white light and exciting light. a, From left to right are mCherry, eGFP, and blank cells. These cells did not show apparent difference under white light. b, Florescent cells and blank cells exposed to exciting light.]]
+
+
To demonstrate that the software was able to accurately measure fluorescent intensity of tattoo cells, we diluted mCherry-293 cells to a panel of concentrations by mixing with blank cells (Figure. 3), and made a standard curve (Figure. 4). The percentage of mCherry-293 cells in diluted cells were 20%, 40%, 60%, 80%, and 100%, respectively. The standard samples were taken photos by a camera and their fluorescence intensity was measured by using the software to analyze the photos. The standard curve was shown in Figure. 4.
+
[[File:T--SMMU-China--mCherry 3.png|1100px|thumb|center| Figure. 3 Fluorescence of standard samples of different concentrations.]]
+
[[File:T--SMMU-China--mCherry 4.png|600px|thumb|center| Figure. 4 Standard curve of fluorescence intensity of different concentrations]]
+
We also made two samples whose concentrations were 45% and 70%, respectively. Their fluorescence intensity measured by the software were 41.810 and 60.634. Concentrations were calculated by putting the numbers into the standard curve and the results acquired were 44.5% and 69.8%, which were very close to the true value 45% and 70%.
+
+
These results demonstrated fluorescent proteins such as mCherry were sensitive and could be measured quantitively by our software.
+
+
==References==
+
[1] R. Tripathi, “Functional characterisation of LEA proteins from bdelloid rotifers,” 2012.
To enable protein visualization <i>in vivo</i> and <i>in vitro </i> we characterized purified mCherry and fused it to several proteins of interest. We were able to show that neither a fusion at the N-terminus, nor at the C-terminus as well as a double-fusion at the N- and C-terminus severely disturb the ability of mCherry to fluoresce (see parts <a href="https://parts.igem.org/Part:BBa_K2926049">BBa_K2926049</a>, <a href="https://parts.igem.org/Part:BBa_K2926050">BBa_K2926050</a>, <a href="https://parts.igem.org/Part:BBa_K2926051">BBa_K2926051</a> and <a href="https://parts.igem.org/Part:BBa_K2926068">BBa_K2926068</a>) .<br> Additionally, we compared two different purification methods and characterized light-stability and pH sensitivity of mCherry.<br>
+
</div>
+
+
<h2>Usage and Biology</h2>
+
<div class="">
+
Since the first successful cloning of the green fluorescent protein GFP of <i>Aequorea victoria</i> in 1992
+
(Prasher et al. 1992) fluorescent proteins became a widely used tool in many fields of research. In contrast to
+
antibodies labeled with fluorophores that have to cross the cellular membrane which severely disturbes the cellular
+
integrity, flourescing proteins enable live cell imaging and the investigation of native states of the cell.<br>
+
Because of the wide range of applications for fluorescing proteins there was a great interest in finding and engineering
+
improved variants and a wider colour spectrum. In the last few years red fluorescing proteins became more and more
+
important. Common native red fluorescing proteins are often dimeric or tetrameric what makes their usage in experimental
+
setups difficult. Directed mutation of dsRFP from the corallimorpharia <i>Discosoma sp.</i> Led to the first monomeric red
+
fluorescing protein mRFP1 (Shaner et al. 2004). Unfortunately this mutations resulted in a lower quantum yield and
+
decreased photostability (Shaner et al. 2004). During further protein engineering attempts, scientists were able to
+
create the red fluorescent protein mCherry. mCherry is a 26.7 kDa protein that shows a very short maturation time of about
+
15 minutes and a low acid sensitivity. Its excitation maximum lies at 587 nm and it has its emission maxiumum at 610 nm
+
(www.fpbase.org). In 2006 the crystal structure of mCherry was published (Shu and Remington 2006).
<figcaption> <b>Fig. 1: Crystal structure of mCherry.</b><br>
+
</figcaption>
+
</figure>
+
</div>
+
+
mCherry consists of 13 beta-sheets which form a beta-barrel and three alpha helices. The chromophore is made of methionine,
+
tyrosine and glycine who posttranslationally form an imidazolinone (Shu et al. 2006).
+
</div>
+
+
<br> <div style="display: inline-grid;">
+
+
<h2 >Expression</h2>
+
+
+
To further characterize purified mCherry we compared two different purification protocols. Purification
+
via his-tag was compared to the IMPACT-purification protocol from NEB.</div>
+
<div> For this purpose we cloned mCherry (<a href="https://parts.igem.org/Part:BBa_J06504">BBa_J06504</a>) into the purification and expression vector pTXB1 from NEB and at the same time added six
+
histidines to the C-terminus of mCherry in pSB1C3 ( <a href="https://parts.igem.org/Part:BBa_K2926048"> BBa_K2926048 </a> ). Both expression vectors were transformed
+
in <i>E. coli </i> ER2566. After induction with IPTG both cultures showed the characteristic red colour of mCherry
+
expressing bacteria (Fig. 2 and Fig. 3).
<figcaption> <b> Fig. 4: Purification columns for IMPACT- (left) and Ni-TED-purification (right).</b><br>
+
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 000 rpm for 15 s. The lysate
+
was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm. Cleared lysate was loaded onto a chitin column
+
(IMPACT-purification) or a Ni-TED column (purification via his tag) and washed with wash buffer. Finally the protein
+
was eluted, washed in PBS and concentrated.
+
</figcaption>
+
</figure> <br>
+
</div>
+
<div class="">
+
A performed Bradford assay showed that expression and purification using the IMPACT-Kit resulted in a higher yield since
+
we were able to purify 985 µg mCherry from a cell mass of 2.13 g compared to 39.4 µg mCherryHis from a cell mass of 1.92 g.
+
Both purification methods were analyzed on a SDS-PAGE (Fig. 5).
<figcaption> <b> Fig. 8: Fluorescence intensity of the dilution series of the two mCherry variants.</b><br> Fluorescence intensity of
+
a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured
+
(λ<sub>Ex</sub>=570 nm, λ<sub>Em</sub>=610 nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red
+
at the same wavelength.
+
</figcaption>
+
</figure> <br>
+
</div>
+
<div class="">
+
The fluorescence intensity of mCherryHis seemed to be higher than the intensity of mCherry purified via IMPACT kit.
+
This might be due to the different purification protocols. Cleavage of mCherry from the chitin column during the
+
IMPACT-purification is mediated through incubation of the column for 20-24 h in DTT at room temperature. Those
+
purification conditions might have a negative impact on the protein. Compared to Texas Red, the fluorescence intensity of
+
1 µmol mCherryHis equals the fluorescence intensity of 1.92 µmol of the fluorescent dye. In contrast, the fluorescence
+
intensity of 1 µmol mCherry purified via IMPACT protocol equals the fluorescence intensity of 565 nmol Texas Red. <br> <br>
+
Additionally we wanted to characterize the light tolerance and the pH-range of mCherry to get insight into the
+
optimal handling procedures. To determine the stability of mCherry it was exposed to normal daylight in the lab at
+
room temperature for a longer period of time (Fig. 9).
<figcaption> <b> Fig. 11: Fluorescence-spectra of mCherry at pH 6 (pink) and pH 11 (dark purple).</b><br>
+
mCherry was incubated in buffers with different pH for 5 min. The fluorescence spectrum of mCherry at pH 11
+
and pH 6 was measured (λ<sub>Ex</sub>=570 nm, gain calculated from 2.5 µM Texas Red) using
+
the TECAN infinite M200 and normalized to the maximum.
+
</figcaption>
+
</figure> <br>
+
</div>
+
<div class="">
+
The altered fluorescence spectrum of mCherry at pH 11 indicates, that the protein disintegrates at
+
higher pH which somehow results in increased fluorescence intensity. Maybe the altered protein structure
+
exposes the fluorophore to the media which minimizes inner protein quenching.
+
</div>
+
<h2>References</h2>
+
<p> Prasher, D. C.; Eckenrode, V. K.; Ward, W. W.; Prendergast, F. G.; Cormier, M. J. (1992): Primary structure of the Aequorea victoria green-fluorescent protein. In: Gene 111 (2). </p>
+
<p> Shaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y. (2004): Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. In: Nature biotechnology 22 (12).</p>
+
<p> Shu, X.; Remington, S. J. (2006): Crystal structure of mCherry.</p>
+
<p> Shu, Xiaokun; Shaner, Nathan C.; Yarbrough, Corinne A.; Tsien, Roger Y.; Remington, S. James (2006): Novel chromophores and buried charges control color in mFruits. In: Biochemistry 45 (32). </p>
We construct the composite part consisting of two parts: OmpT signal peptide ([[Part:BBa_K3805532]]) and mCherry (BBa_J06504).Outer membrane protease T (OmpT) is a 33.5 kDa endoprotease located on the outer membrane of ''Escherichia coli''.Ompt signal peptide is an excretion tag linked on the N-terminus of proteins.It can transport proteins across the outer membrane with the help of OmpT. In our part, Ompt signal peptide is fused with mCherry, which will enable mCherry to be secreted to extracellular space. For detailed information, please go to part [[Part:BBa_K3805886]]
+
+
== ==
===Functional Parameters===
===Functional Parameters===
<partinfo>BBa_J06504 parameters</partinfo>
<partinfo>BBa_J06504 parameters</partinfo>
<!-- -->
<!-- -->
Latest revision as of 23:18, 13 October 2022
monomeric RFP optimized for bacteria
mRFP1-derived, altered to be a BioBrick by removing a PstI site and adding BioBrick ends. [mRFP1 was itself a derived from DsRed (via 33 mutations!)]
mCherry is one of several "second-generation" monomeric fluorescent proteins developed in Roger Tsien's laboratory at UCSD (cf., Nature Biotechnology22, 1567 - 1572 (2004). PMID 15558047
Usage and Biology
Some strange experimental results that have been seen could be explained by an internal RBS + start. The 10th amino acid is a Met which is preceded by AGGAGGA(NNNN). This is almost a perfect consensus RBS so it seems quite likely that translation can begin 10 amino acids in. Note that mCherry was designed by fusing the N and C terminal regions of EGFP on to a mRFP variant (to increase tolerance to protein fusions). Thus, removing the first several amino acids is not expected to have much effect on fluorescence. If this is truly a strong internal RBS, then the identity of any attached RBS may have little effect. Also, one should be careful when making protein fusions. --Austin
The copy as provided in the 2010 distribution is incorrect - it contains ~500 bp of something that is not mCherry between the VF2 and VR sites. You can get a functioning copy via PCR out of Part:BBa_J06702.
--[http://openwetware.org/wiki/User:Joseph_T._Meyerowitz jmeyerow]
Improvement by SYSU-CHINA 2016
This part has been improved to be the mCHERRY UNIT including a degradation tag named DBOX, a fluorescent gene mCherry and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. The whole part can be cut off by VIKA.
For more information of the improved part, please go to the page of Part:BBa_K1926013.
Improvement by USP_UNIFESP-Brazil
The reporter mCherry has been codon-optimized for Chlamydomonas reinhardtii and it was successfully tested as a reporter in this chassis. We have made several measurements to show its fluorescence in Chlamydomonas reinhardtii as well its properties (Fluorescence excitation/emission spectrum). For more information of the improved part, please go to the page of Part:BBa_K2136016.
The internal RBS-like sequence in this mCherry reporter was removed, and the overall sequence was codon-optimized for E. coli K12. This new part BBa_K2375001 should be more suitable for creating fusion proteins.
Improvement by TAU_Israel 2021
The codon sequence has been optimized for expression in Bacillus subtilis against E. coli using the Communique tool using several methods. For more information, refer to part pages BBa_K3871001, BBa_K3871002, BBa_K3871003, BBa_K3871004,BBa_K3871005 and the 2021 TAU_Israel wiki, available here.
Improvement by UCopenhagen 2022
We improved this mCherry protein by adding SnoopCatcher (part BBa_K4247009), so that it can now spontaneously form an irreversible isopeptide bond with any protein that contains the SnoopTag (part BBa_K4247008). By adding a functional SnoopCatcher to mCherry and validating the SnoopTag-Catcher system, we have successfully improved the part BBa_J06504. Thus, mCherry with the SnoopCatcher can bind to essentially any protein with a SnoopTag. Since mCherry is a widely used protein, we believe our improvement opens up many new uses of the protein among its repertoire of diverse biotechnological applications. For more information about our improved part, go to BBa_K4247025.
Contribution: TU_Eindhoven 2019
In this contribution, we futher characterized mCherry (BBa_J06504). mCherry was cloned into a pET28a(+) vector (containing an N-terminal 6xHis-tag) and subsequently expressed in BL21 (DE3) E. coli and purified using immobilized metal affinity chromatography (IMAC). Different concentrations of mCherry were made and an increase in color intensity was visible (Figure 1).
Figure 1. Purified mCherry in different concentrations, showing a clear increase in color intensity.
Consequently, the purified protein was analyzed on an SDS-PAGE (Figure 2) which shows a clear blob above 25 kDa, corresponding with 6xHis-tagged mCherry’s molecular weight of 29.3 kDa. The blob in between 10 kDa and 15 kDa is a truncated version of mCherry as described by Tripathi [1]. There are other bands visible so the purity is not optimal. However, the contrast between the huge blobs and the other bands is very big.
Figure 2. SDS-PAGE of mCherry after purification.
Following protein purification, the protein functionality was analyzed. Firstly, the excitation and emission of mCherry were measured within its absorption and emission range (Figure 3). This shows a clear excitation maximum at 587 nm and an emission maximum at 610 nm.
Secondly, the photostability of mCherry was measured following UV radiation up until thirty minutes to advance usage of mCherry in experiments (Figure 4). Exposure to UV light shows a decrease in fluorescence intensity. After 30 min, the intensity is almost halved compared to no UV exposure.
Figure 3. Excitation and emission spectrum of mCherry.AAAAAAAAAAFigure 4. Excitation spectrum of mCherry after different periods of UV radiation.
HUBU-WUHAN 2019-Characterization
We characterized the bleaching effect in zymomonas mobilis using fluorescent microscopy Leica DMi8. We obtained image information through fluorescence microscope and analyzed it with the Rleative Flourescense Calculator in LAS X.
GIF.1 Real-time imaging of zymomonas mobilis.
It is expressed in pEZ-15A plasmid with a PlacUV5 promoter. LAS Z was used to analyze the images and calculate the rate of photobleaching. We obtained a half-life period as t1/2=40.07s
Fig.1 Curve fit of relative intensity.
Characterization: SMMU-China 2019
In our project this year, we aim to develop a biomedical tattoo that could detect disease related molecules and output signals through accumulation of fluorescent proteins. We have also developed a software to analyze the intensity of the fluorescence so that this signal could reflect the disease conditions. To test whether this software could accurately measure the fluorescence and whether florescent proteins are sensitive enough to meet our conception, we carried out the following experiments:
First, we cloned this part (BBa_J06504) into eukaryotic expression vector pcDNA3.1 and transfected it into HEK293T cells through Lipofectamine 2000 transfection reagent. Twenty-four and 48 hours after transfection, bright red light could be observed under florescence microscopy (Figure. 1)
Figure. 1 mCherry expression at 24 and 48 hours after transfection.
The transfected and untransfected HEK293T cells were digested at 24h by trypsin and centrifugated in microtubes. The cells in microtubes were used to mimic the biomedical tattoo and were further examined. Whereas the cells did not show apparent difference with negative control under white light (Figure. 2a), they emitted red light when exposed to exciting light from a mercury lamp (Figure. 2b). This result suggested that fluorescent proteins were sensitive reporters and could be distinguished even by naked eyes under exciting light.
Figure. 2 Tattoo cells under white light and exciting light. a, From left to right are mCherry, eGFP, and blank cells. These cells did not show apparent difference under white light. b, Florescent cells and blank cells exposed to exciting light.
To demonstrate that the software was able to accurately measure fluorescent intensity of tattoo cells, we diluted mCherry-293 cells to a panel of concentrations by mixing with blank cells (Figure. 3), and made a standard curve (Figure. 4). The percentage of mCherry-293 cells in diluted cells were 20%, 40%, 60%, 80%, and 100%, respectively. The standard samples were taken photos by a camera and their fluorescence intensity was measured by using the software to analyze the photos. The standard curve was shown in Figure. 4.
Figure. 3 Fluorescence of standard samples of different concentrations.
Figure. 4 Standard curve of fluorescence intensity of different concentrations
We also made two samples whose concentrations were 45% and 70%, respectively. Their fluorescence intensity measured by the software were 41.810 and 60.634. Concentrations were calculated by putting the numbers into the standard curve and the results acquired were 44.5% and 69.8%, which were very close to the true value 45% and 70%.
These results demonstrated fluorescent proteins such as mCherry were sensitive and could be measured quantitively by our software.
References
[1] R. Tripathi, “Functional characterisation of LEA proteins from bdelloid rotifers,” 2012.
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]
Contribution: iGEM Bielefeld-CeBiTec 2019
To enable protein visualization in vivo and in vitro we characterized purified mCherry and fused it to several proteins of interest. We were able to show that neither a fusion at the N-terminus, nor at the C-terminus as well as a double-fusion at the N- and C-terminus severely disturb the ability of mCherry to fluoresce (see parts BBa_K2926049, BBa_K2926050, BBa_K2926051 and BBa_K2926068) . Additionally, we compared two different purification methods and characterized light-stability and pH sensitivity of mCherry.
Usage and Biology
Since the first successful cloning of the green fluorescent protein GFP of Aequorea victoria in 1992
(Prasher et al. 1992) fluorescent proteins became a widely used tool in many fields of research. In contrast to
antibodies labeled with fluorophores that have to cross the cellular membrane which severely disturbes the cellular
integrity, flourescing proteins enable live cell imaging and the investigation of native states of the cell.
Because of the wide range of applications for fluorescing proteins there was a great interest in finding and engineering
improved variants and a wider colour spectrum. In the last few years red fluorescing proteins became more and more
important. Common native red fluorescing proteins are often dimeric or tetrameric what makes their usage in experimental
setups difficult. Directed mutation of dsRFP from the corallimorpharia Discosoma sp. Led to the first monomeric red
fluorescing protein mRFP1 (Shaner et al. 2004). Unfortunately this mutations resulted in a lower quantum yield and
decreased photostability (Shaner et al. 2004). During further protein engineering attempts, scientists were able to
create the red fluorescent protein mCherry. mCherry is a 26.7 kDa protein that shows a very short maturation time of about
15 minutes and a low acid sensitivity. Its excitation maximum lies at 587 nm and it has its emission maxiumum at 610 nm
(www.fpbase.org). In 2006 the crystal structure of mCherry was published (Shu and Remington 2006).
Fig. 1: Crystal structure of mCherry.
mCherry consists of 13 beta-sheets which form a beta-barrel and three alpha helices. The chromophore is made of methionine,
tyrosine and glycine who posttranslationally form an imidazolinone (Shu et al. 2006).
Expression
To further characterize purified mCherry we compared two different purification protocols. Purification
via his-tag was compared to the IMPACT-purification protocol from NEB.
For this purpose we cloned mCherry (BBa_J06504) into the purification and expression vector pTXB1 from NEB and at the same time added six
histidines to the C-terminus of mCherry in pSB1C3 ( BBa_K2926048 ). Both expression vectors were transformed
in E. coli ER2566. After induction with IPTG both cultures showed the characteristic red colour of mCherry
expressing bacteria (Fig. 2 and Fig. 3).
Fig 2: Expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).
Expression cultures of mCherry in pTXB1 and mCherryHis in pSB1C3 in E. coli ER2566 were cultivated to an OD of
around 0.6 at 37 °C in LB with 100 mg Ampicillin per L. Protein expression was induced by addition of IPTG to a final
concentration of 0.4 mM. After additional 30 min at 37 °C cultures were transferred to 17 °C and protein expression
was performed over night.
The expression culture of mCherry in pTXB1 showed a brighter red colour which indicated a higher expression level.
Fig. 3: Harvested expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).
Expression cultures of mCherry and mCherryHis in E. coli ER2566 were harvested by centrifugation at
4 °C for 20 min and 4 000 rpm.
Purification
After cultivation and cell lysis via Ribolyzer the protein was purified using the His-purification kit from Macherey Nagel
and the IMPACT-purification kit from NEB (Fig. 4).
Fig. 4: Purification columns for IMPACT- (left) and Ni-TED-purification (right).
Harvested cells were lysed using Zirconia metal beads (1 mm) in a Ribolyzer at 8 000 rpm for 15 s. The lysate
was cleared by centrifugation at 4 °C for 1 h and 4 500 rpm. Cleared lysate was loaded onto a chitin column
(IMPACT-purification) or a Ni-TED column (purification via his tag) and washed with wash buffer. Finally the protein
was eluted, washed in PBS and concentrated.
A performed Bradford assay showed that expression and purification using the IMPACT-Kit resulted in a higher yield since
we were able to purify 985 µg mCherry from a cell mass of 2.13 g compared to 39.4 µg mCherryHis from a cell mass of 1.92 g.
Both purification methods were analyzed on a SDS-PAGE (Fig. 5).
Fig. 5: SDS-PAGE of the protein purification. E. coli lysate of the expression culture,
flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98 °C
for 10 min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12 %). The proteins
were separated through electrophoresis (25 mA). Suggested mCherry bands in the lane with purified proteins were
marked in dark red.
The SDS-PAGE shows an intense band at the estimated height of around 27 kDa in every lane. This indicates that mCherry
as well as mCherryHis have successfully been expressed. The bands in the wash- and flow-through-fraction show, that not
all of the protein efficiently binds to the purification columns.
In the last lane you can see that we were able to purify mCherry as well as mCherryHis. While the IMPACT-purification
resulted in a higher yield, the purity of mCherryHis was higher as the protein lane in Fig. 5 indicated.
Following the SDS-PAGE we analyzed the purified protein via MALDI-ToF. For this purpose we excised the marked bands (Fig. 5)
from the SDS-PAGE and started a tryptic digestion of the washed gel fragment. Analysis via MALDI-ToF confirmed that we were
able to purify mCherry (Fig. 6).
Fig. 6: Mass spectrum of mCherry (1) and mCherryHis (2) after tryptic digestion.
Excised bands from the SDS-PAGE of mCherry and mCherryHis were washed, digested over night with trypsine and
co-cristallyzed with a HCCA-matrix on a MALDI target. Mass spectrum was recorded in a MALDI-ToF MS from Bruker
Daltronics and data was evaluated using the software BioTools.
The generated mass spectra and mass lists were evaluated using the software BioTools. To compare the experimentally
determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected protein
sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match both proteins
to the theoretical spectrum. Additionally we were able to detect the his-tag from mCherryHis in the mass list.
Characterization
To gain some more knowledge about mCherry we analyzed different properties of the protein. First of all we measured its
emission- and excitation spectra (Fig. 7).
Fig. 7: Emission- and excitation spectra of mCherry. Emission- (dashed lines) and excitation-spectra
(solid lines) of mCherry purified via IMPACT-Kit (dark purple) and His-tag (pink) were measured (λEx=570 nm,
λEm=600 nm to 850 nm) using the TECAN infinite M200 and normalized to their maximum.
The resulting spectra show, that adding a his-tag to mCherry does not alter the emission- or excitation spectrum of mCherry.
The excitation maximum of mCherry lies at 587 nm, the emission maximum at 608 nm.
Next we compared the fluorescence intensity of the two different mCherry-variants normalized to Texas Red (Fig. 8).
Fig. 8: Fluorescence intensity of the dilution series of the two mCherry variants. Fluorescence intensity of
a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured
(λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red
at the same wavelength.
The fluorescence intensity of mCherryHis seemed to be higher than the intensity of mCherry purified via IMPACT kit.
This might be due to the different purification protocols. Cleavage of mCherry from the chitin column during the
IMPACT-purification is mediated through incubation of the column for 20-24 h in DTT at room temperature. Those
purification conditions might have a negative impact on the protein. Compared to Texas Red, the fluorescence intensity of
1 µmol mCherryHis equals the fluorescence intensity of 1.92 µmol of the fluorescent dye. In contrast, the fluorescence
intensity of 1 µmol mCherry purified via IMPACT protocol equals the fluorescence intensity of 565 nmol Texas Red.
Additionally we wanted to characterize the light tolerance and the pH-range of mCherry to get insight into the
optimal handling procedures. To determine the stability of mCherry it was exposed to normal daylight in the lab at
room temperature for a longer period of time (Fig. 9).
Fig. 9: Fluorescence intensity of mCherry in dependence on light exposure. mCherry was exposed to normal
daylight at room temperature. The remaining fluorescence intensity was measured at determinated time points (λEx=570 nm,
λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the intensity at t=0.
The results show that there is no significant decrease in the fluorescence intensity within the first 30 minutes.
After 2 h the fluorescence intensity already decreases more than 50 %.
An often stated advantage of mCherry is its low acid sensitivity. To analyze the pH-range of mCherry we
measured the remaining fluorescence intensity after incubation in buffers with different pH (Fig. 10).
Fig. 10: Fluorescence intensity of mCherry in dependence on the pH. mCherry was incubated for
5 minutes in buffers with different pH. The remaining fluorescence intensity was measured (λEx=570 nm,
λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the intensity at pH 7.
Detectable fluorescence could be measured in a pH-range from ph 4 to pH 12 while the pH-optimum is 6-7.
Interestingly the fluorescence intensity seems to have a second optimum at pH 10-11. To verificate this we
measured the fluorescence-spectra of mCherry at pH 6 and pH 11 and compared them (Fig. 11).
Fig. 11: Fluorescence-spectra of mCherry at pH 6 (pink) and pH 11 (dark purple).
mCherry was incubated in buffers with different pH for 5 min. The fluorescence spectrum of mCherry at pH 11
and pH 6 was measured (λEx=570 nm, gain calculated from 2.5 µM Texas Red) using
the TECAN infinite M200 and normalized to the maximum.
The altered fluorescence spectrum of mCherry at pH 11 indicates, that the protein disintegrates at
higher pH which somehow results in increased fluorescence intensity. Maybe the altered protein structure
exposes the fluorophore to the media which minimizes inner protein quenching.
References
Prasher, D. C.; Eckenrode, V. K.; Ward, W. W.; Prendergast, F. G.; Cormier, M. J. (1992): Primary structure of the Aequorea victoria green-fluorescent protein. In: Gene 111 (2).
Shaner, Nathan C.; Campbell, Robert E.; Steinbach, Paul A.; Giepmans, Ben N. G.; Palmer, Amy E.; Tsien, Roger Y. (2004): Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. In: Nature biotechnology 22 (12).
Shu, X.; Remington, S. J. (2006): Crystal structure of mCherry.
Shu, Xiaokun; Shaner, Nathan C.; Yarbrough, Corinne A.; Tsien, Roger Y.; Remington, S. James (2006): Novel chromophores and buried charges control color in mFruits. In: Biochemistry 45 (32).
Inprovement by BNU-China 2021
edited by Qiuchen Gu
We construct the composite part consisting of two parts: OmpT signal peptide (Part:BBa_K3805532) and mCherry (BBa_J06504).Outer membrane protease T (OmpT) is a 33.5 kDa endoprotease located on the outer membrane of Escherichia coli.Ompt signal peptide is an excretion tag linked on the N-terminus of proteins.It can transport proteins across the outer membrane with the help of OmpT. In our part, Ompt signal peptide is fused with mCherry, which will enable mCherry to be secreted to extracellular space. For detailed information, please go to part Part:BBa_K3805886
