Difference between revisions of "Part:BBa K2842669"

Revision as of 02:31, 18 October 2018

mScarlet reporter with TerL-C intein on the N terminus

mScarlet with TerL-C intein
Function	Functionalise and cap inteins polymers
Use in	E. coli cells
Chassis Tested	DH5α cells
Abstraction Hierarchy	Composite Device
Related Device	BBa_K1362101
RFC standard	RFC10,RFC21,RFC23 & RFC25 compatible
Backbone	pSB1C3
Submitted by	[http://2018.igem.org/Team:UCL UCL iGEM 2018]

This DNA construct encodes a C-terminal segment of the AceL-TerL intein fused to the N-terminus of a mScarlet reporter protein which contains a C-terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between mScarlet and another protein bound to a complementary split intein. During this process the intein splices itself out of the protein and is not present in the final fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter. BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N-terminal intein segment to enable intein trans-splicing.

Discovered in the late 1980s, inteins arewere found to be naturally occurring protein segments attached to specific host proteins of unicellular organisms (Protein Engineering Handbook, 2009). Split-inteins are expressed in two separate proteins as N or C terminal domains. Either half can be bound to unique external proteins to create intein fusion proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction (depicted in Figure 1), which allows for the ligation of the external proteins through a peptide bond.

Figure 1: Protein trans-splicing This image depicts the ability of split inteins to form pepide bonds between two proteins of interest while splicing themselves ut of the end product.Inteins ligate their flanking sequences with a native peptide bond. These sequences can be either their native exteins or unrelated peptides or proteins. Figure adapted from Thiel et al., 2014 [1]

BBa_K2842669 utilises an AceL-TerL-C split intein, which are complementary to one of the the split inteins on BBa_K2842680. AceL-TerL-C is derived from phage genes discovered in antarctic permanently stratified saline lakes. It’s corresponding N terminal intein is the smallest N-terminal split intein as it is composed of only 25 amino acids [1].

Reporter Protein BBa_K2842669 encodes the reporter protein, mScarlet, which is a highly engineered monomeric red fluorescent protein.

Experimental Results

Figure 1: Comparisons of fluorescence/OD600 of BBa_K2842669 and BBa_K1362101

A plate reader was used to measure the fluorescence of whole cell cultures.Plate reader settings: Filters were set to 540/590 , shaking at 200 rpm for 20 h at 25oC. (1-Red) BBa_K2842669 in BL21*(DE3) cells induced with 400 μM IPTG
(2-Orange) BBa_K2842669 in BL21*(DE3) cells without IPTG induction
(3-Grey) BBa_K1362101 expressed in BL21*(DE3) cells />

This construct (BBa_K2842669) is also an improvement of the part BBa_K1362101. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but our part has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 [2][3]. Our reporter protein mScarlet has an 11-fold increase in fluorescence/OD600 compared to BBa_K1362101 when driven by the expression from the T7 promoter (Fig3). Our construct also has a dual function as both an assembly construct and reporter for building new inteins, and as an intein fusion by itself. Due to this, it can be used both to test intein functionality in other constructs and to build new ones.

We measured the expression of BBa_K2842669 with a BMG Fluostar plate reader by analysis of its fluorescence at varying IPTG concentrations. We found that mScarlet is very well expressed when produced from an IPTG-inducible T7 promoter. Increasing IPTG concentrations from 400 μM to 800 μM had little effect on expression levels and cell growth rate (Fig1A, Fig1B), indicating that IPTG concentrations at 400 μM are sufficient to overproduce our protein. We also tested the modularity of the construct by replacing mScarlet with with two new sequences to create the biobricks BBa_K2842710 and BBa_K2842720. Diagnostic digests with BsaI ((Figure 2) and the sequencing results confirmed that the cloning was successful.

Figure 2: Expression of IP at varying IPTG concentrations A plate reader was used to measure the fluorescence of whole cell cultures after induction with various concentrations of ITPG. Plate reader settings: Filters 540/590 , shaking at 200 rpm for 20 h at 25oC. This graph shows the fold change in fluorescence of BBa_K2832669 at a range of IPTG induction concentrations relative to uninduced BBa_K2832669. </p>

Figure 3: SDS-PAGE of BBa_K2832669

We expressed BBa_K2832669 in 50 mL cultures and extracted the intein fusion protein through sonication. The clear lysate was purified on Strep-Tactin columns then SDS-PAGE was performed on the samples. 3A shows the SDS gel when stained with instant blue for 6 hours. 3B shows the fluorescence reading from the gel. (L) Protein standard numbers are in kDa (1) The clear lysate of BBa_K2832669 before purification (2) After purification on Streptactin columns

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 941
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1224
Illegal BsaI.rc site found at 28
Illegal SapI site found at 1130
Illegal SapI.rc site found at 419

Functional Parameters

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 10: (underlined part encodes the protein)
GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCGCAAATTTAAGGATTCTCAAAAATGTTCCGC ... TTCGAAAAA
TGAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA
ORF from nucleotide position 96 to 1166 (excluding stop-codon)

Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

1
101
201
301

MFRTNTNNIKILSPNGFSNFNGIQKVERNLYQHIIFDDDTEIKTSIDHPFGKDKIQARDVKVGDYLNSKKVLYNELVNENIFLYDPINVEKESLYITNGV
VSHNCYNGGRASMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSF
PEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPV
QMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYKGSSSEWSHPQFEK*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

	RFC25 scar (shown in bold):	336 to 337
	Strep-tag II:	350 to 357

Amino acid composition:

Ala (A)	12 (3.4%)
Arg (R)	15 (4.2%)
Asn (N)	20 (5.6%)
Asp (D)	23 (6.4%)

Cys (C)	1 (0.3%)
Gln (Q)	11 (3.1%)
Glu (E)	29 (8.1%)
Gly (G)	33 (9.2%)

His (H)	9 (2.5%)
Ile (I)	18 (5.0%)
Leu (L)	20 (5.6%)
Lys (K)	32 (9.0%)

Met (M)	12 (3.4%)
Phe (F)	18 (5.0%)
Pro (P)	17 (4.8%)
Ser (S)	22 (6.2%)

Thr (T)	21 (5.9%)
Trp (W)	4 (1.1%)
Tyr (Y)	18 (5.0%)
Val (V)	22 (6.2%)

Amino acid counting

	Total number:	357
	Positively charged (Arg+Lys):	47 (13.2%)
	Negatively charged (Asp+Glu):	52 (14.6%)
	Aromatic (Phe+His+Try+Tyr):	49 (13.7%)

Biochemical parameters

	Atomic composition:	C₁₈₁₄H₂₇₈₃N₄₈₇O₅₅₄S₁₃
	Molecular mass [Da]:	40694.8
	Theoretical pI:	6.05
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	48820 / 48883 (all Cys red/ox)

Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.73)	good (0.68)	acceptable (0.56)	good (0.65)	excellent (0.85)	excellent (0.86)

Alignments (obtained from PredictProtein.org)
There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.

Predictions (obtained from PredictProtein.org)

There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.

The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
If you have any questions, comments or suggestions, please leave us a comment.

References

[1] Thiel IV, Volkmann G, Pietrokovski S and Mootz HD. (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Communications, Int. Ed., 53: 1306-1310.
[2] Bindels DS, Haarbosch L, van Weeren L, Postma M, Wieser KE, Mastop M, et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods. 2017;14(1):53-6.
[3] Robert E. Campbell, Oded Tour, Amy E. Palmer, Paul A. Steinbach, Geoffrey S. Baird, David A. Zacharias, Roger Y. Tsien, A monomeric red fluorescent protein Proceedings of the National Academy of Sciences Jun 2002, 99 (12) 7877-7882

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 |'''Function'''
-|Standardised blue-white screening
+|Functionalise and cap inteins polymers
 |-
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-====References====
-<p>
-[1] Thiel IV, Volkmann G, Pietrokovski S and Mootz HD. (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Communications, Int. Ed., 53: 1306-1310.
-<br>
-[2] Bindels DS, Haarbosch L, van Weeren L, Postma M, Wieser KE, Mastop M, et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods. 2017;14(1):53-6.
-<br>
-[3] Robert E. Campbell, Oded Tour, Amy E. Palmer, Paul A. Steinbach, Geoffrey S. Baird, David A. Zacharias, Roger Y. Tsien, A monomeric red fluorescent protein Proceedings of the National Academy of Sciences Jun 2002, 99 (12) 7877-7882
-</p>
@@ Line 181: / Line 170: @@
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font-family:Arial,Helvetica,sans-serif; background:rgb(255, 200, 0); background-color:rgba(255, 200, 0, 1); height:16px; width:16px; top:80px; border-radius:50%; } </style><div id='AutoAnnotator_container_1539812017099'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1539812017099 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_<!------------------------Enter BioBrick number here------------------------>">BBa_<!------------------------Enter BioBrick number here------------------------></a> automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCGCAAATTTAAGGATTCTCAAAA<u>ATGTTCCGC&nbsp;...&nbsp;TTCGAAAAA</u><br>TGAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 96 to 1166 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;<br>301&nbsp;</td><td class="AutoAnnotatorSeqSeq">MFRTNTNNIKILSPNGFSNFNGIQKVERNLYQHIIFDDDTEIKTSIDHPFGKDKIQARDVKVGDYLNSKKVLYNELVNENIFLYDPINVEKESLYITNGV<br>VSHNCYNGGRASMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSF<br>PEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPV<br>QMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS<b>TG</b>GMDELYKGSSSE<u>WSHPQFEK</u>*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2a">RFC25 scar (shown in bold):&nbsp;</td><td class="AutoAnnotatorSeqFeat3">336 to 337</td></tr><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2a"><i>Strep</i>-tag II:&nbsp;</td><td class="AutoAnnotatorSeqFeat3">350 to 357</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid composition:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Ala (A)</td><td class="AutoAnnotatorInnerAmino">12 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">15 (4.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">20 (5.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">23 (6.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">1 (0.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">11 (3.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">29 (8.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">33 (9.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">9 (2.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">18 (5.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">20 (5.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">32 (9.0%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">12 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">18 (5.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">17 (4.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">22 (6.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">21 (5.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">4 (1.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">18 (5.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">22 (6.2%)</td></tr></table></td></tr></table></td></tr><tr><td class="AutoAnnotatorAminoCountingOuter"><strong>Amino acid counting</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Total number:</td><td class="AutoAnnotatorAminoCountingInner3">357</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">47 (13.2%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">52 (14.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">49 (13.7%)</td></tr></table></td><td class="AutoAnnotatorBiochemParOuter"><strong>Biochemical parameters</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Atomic composition:</td><td class="AutoAnnotatorBiochemParInner3">C<sub>1814</sub>H<sub>2783</sub>N<sub>487</sub>O<sub>554</sub>S<sub>13</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">40694.8</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">6.05</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Extinction coefficient at 280 nm [M<sup>-1</sup> cm<sup>-1</sup>]:</td><td class="AutoAnnotatorBiochemParInner3">48820 / 48883 (all Cys red/ox)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges</strong>&nbsp;<input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1539812017099()' value='Show'><span id="hydrophobicity_charge_explanation"></span><div id="hydrophobicity_charge_container" style='display:none'><div id="hydrophobicity_charge_placeholder0" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder1" style="width:100%;height:150px"></div></div></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Codon usage</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Organism:</td><td class="AutoAnnotatorCodonUsage3"><i>E. coli</i></td><td class="AutoAnnotatorCodonUsage3"><i>B. subtilis</i></td><td class="AutoAnnotatorCodonUsage3"><i>S. cerevisiae</i></td><td class="AutoAnnotatorCodonUsage3"><i>A. thaliana</i></td><td class="AutoAnnotatorCodonUsage3"><i>P. patens</i></td><td class="AutoAnnotatorCodonUsage3">Mammals</td></tr><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):</td><td class="AutoAnnotatorCodonUsage3">good (0.73)</td><td class="AutoAnnotatorCodonUsage3">good (0.68)</td><td class="AutoAnnotatorCodonUsage3">acceptable (0.56)</td><td class="AutoAnnotatorCodonUsage3">good (0.65)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.85)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.86)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Alignments</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)<br>&nbsp;&nbsp;&nbsp;There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.</td></tr><tr><th id='AutoAnnotatorHeader' colspan="2"><strong>Predictions</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)</th></tr><tr><td class="AutoAnnotator1col" colspan="2">&nbsp;&nbsp;&nbsp;There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.</td><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="http://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software">documentation</a>.<br>If you have any questions, comments or suggestions, please leave us a <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table></div><br><!-- IMPORTANT: DON'T REMOVE THIS LINE, OTHERWISE NOT SUPPORTED FOR IE BEFORE 9 --><!--[if lte IE 8]><script language="javascript" type="text/javascript" src="http://2013.igem.org/Team:TU-Munich/excanvas.js"></script><![endif]--><script type='text/javascript' src='http://code.jquery.com/jquery-1.10.0.min.js'></script><script type='text/javascript' src='http://2013.igem.org/Team:TU-Munich/Flot.js?action=raw&ctype=text/js'></script><script>var jqAutoAnnotator = jQuery.noConflict(true);function show_or_hide_plot_1539812017099(){hydrophobicity_datapoints = 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){jqAutoAnnotator('#AutoAnnotator_container_1539812017099 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1539812017099 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for hydrophobicity (<img src=\'https://static.igem.org/mediawiki/2013/e/e9/TUM13_hydrophobicity_icon.png\' alt=\'blue graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for charge (<img src=\'https://static.igem.org/mediawiki/2013/3/3e/TUM13_charge_icon.png\' alt=\'red graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'>&nbsp;Predicted disulfid bridges (<img src=\'https://static.igem.org/mediawiki/2013/2/28/TUM13_dis_icon.png\' alt=\'yellow circle\' height=\'10\'></img>) with the number of the bridge in the center';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'>&nbsp;Predicted transmembrane helices (<img src=\'https://static.igem.org/mediawiki/2013/7/78/TUM13_trans_icon.png\' alt=\'turquois bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'>&nbsp;Predicted secondary structure: Helices (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_helix_icon.png\' alt=\'violet bars\' height=\'10\'></img>) and beta-strands (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_strand_icon.png\' alt=\'yellow bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' 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+====References====
+<p>
+[1] Thiel IV, Volkmann G, Pietrokovski S and Mootz HD. (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Communications, Int. Ed., 53: 1306-1310.
--
+<br>
+[2] Bindels DS, Haarbosch L, van Weeren L, Postma M, Wieser KE, Mastop M, et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods. 2017;14(1):53-6.
+<br>
-<!-- Add more about the biology of this part here
+[3] Robert E. Campbell, Oded Tour, Amy E. Palmer, Paul A. Steinbach, Geoffrey S. Baird, David A. Zacharias, Roger Y. Tsien, A monomeric red fluorescent protein Proceedings of the National Academy of Sciences Jun 2002, 99 (12) 7877-7882
-===Usage and Biology===
+</p>