Revision as of 13:22, 15 October 2016

Eukaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)

CRY2
Function	blue light stimulated photoreceptor
RFC standard	RFC 10
Backbone	pSB1C3
Organism	Arabidopsis thaliana
Source	Arabidopsis thaliana
Submitted by	[http://2016.igem.org/Team:NEU-China NEU-China 2016]

Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].

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)
ATGAAGATG ... GGTTGCAAATAATAA
ORF from nucleotide position 1 to 1836 (excluding stop-codon)

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

1
101
201
301
401
501
601

MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVF
NHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEK
PSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNCTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYI
CFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYI
SGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPD
EIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSD
QITTSLGKNGCK*

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

None of the supported features appeared in the sequence

Amino acid composition:

Ala (A)	47 (7.7%)
Arg (R)	33 (5.4%)
Asn (N)	21 (3.4%)
Asp (D)	35 (5.7%)

Cys (C)	12 (2.0%)
Gln (Q)	18 (2.9%)
Glu (E)	48 (7.8%)
Gly (G)	36 (5.9%)

His (H)	13 (2.1%)
Ile (I)	36 (5.9%)
Leu (L)	59 (9.6%)
Lys (K)	38 (6.2%)

Met (M)	12 (2.0%)
Phe (F)	25 (4.1%)
Pro (P)	31 (5.1%)
Ser (S)	54 (8.8%)

Thr (T)	24 (3.9%)
Trp (W)	22 (3.6%)
Tyr (Y)	17 (2.8%)
Val (V)	31 (5.1%)

Amino acid counting

	Total number:	612
	Positively charged (Arg+Lys):	71 (11.6%)
	Negatively charged (Asp+Glu):	83 (13.6%)
	Aromatic (Phe+His+Try+Tyr):	77 (12.6%)

Biochemical parameters

	Atomic composition:	C₃₁₂₅H₄₈₁₄N₈₃₆O₉₁₃S₂₄
	Molecular mass [Da]:	69473.1
	Theoretical pI:	5.69
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	146330 / 147080 (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 (0.69)	good (0.73)	good (0.69)	good (0.78)	good (0.80)	good (0.67)

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.

Proof of function

Verification of the suppression efficiency of gRNA
With (BBa_K1982006) different target loci have been tested by the usage of a GFP reporter plasmid(pCold-1) with a CSPA promotor. The target sites can be determined by directing the gRNA consisting of 20 bp length against the desired sequence of interest. R1 with target sites at different distances to the promotor regions proved successfully as potential activation sites (see Table 1 and Figure 3).

Name	Binding Site	Distance to promoter	Sequence	Position
F1	CSPA promoter	15	TGCATCACCCGCCAATGCG	sense sequences
F2	non-coding	68	GCCGCCGCAAGGAATGGTG	sense sequences
R1	CSPA promoter	43	ATTAATCATAAATATGAAA	antisense sequences
R2	non-coding	94	CATCATCCAACTCCGGCAAC	antisense sequences

Table 1: Overview of the tested gRNAs with different binding sites on the GFP pCold-1 plasmid.

Figure 3: Position of the target loci on the GFP pCold-1 plasmid.

To ensure the suppression efficiency of the gRNA, four gRNA sequences targeting different sites of CSPA promoter were designed and transfected into the E. coli strains BL21. Efficient suppression of CSPA promoter in strains BL21 was observed. GFP levels in GFP transgenic strains decrease after inserting a fragment that expresses CSPA promoter gRNA. And the sequence with the best suppression effect was selected for further study.

Figure 4: Results of the GFP-influence under the CSPA promotor
only using different gRNAs targeted to CSPA promoter in BL21..
1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) 9:Control group transfected with GFP plasmid,~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8) at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Silencing capability validation

We next evaluated the effect of tCas9-cibn on suppressing CSPA promoter. GFP expression levels were assayed in strains BL21 after co-transformation with tCas9-cibn and CRY2-VP64 and gRNA in the absence of light conditions.

Figure 5: Silencing capability of tCas9-CIBN with gRNA
using different gRNAs targeted to CSPA promoter in BL21.
1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) and tCas9-CIBN plasmid(pBAD promoter)
9 10:Control groups transfected with GFP plasmid and tCas9-CIBN plasmid(pBAD promoter), ~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8 10) using 15mM L-arabinose at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Compared with control groups, green fluorescence intensity and mRNA levels were dramatically reduced in groups treated with gRNA and tCas9-cibn. These results suggest that gRNA can specifically guide tCas9 to target upstream of CSPA promoter, thereby to inhibit CSPA promoter to reduce GFP expression levels.

Activation of a fluorescence reporter

Spatially controlled activation of gene expression was achieved in strains co-transfected with the LACE system, a reporter vector containing a gRNA target sequence upstream of CSPA promoter and the eGFP gene. Strains transfected with light-activated CRISPR/Cas9 effector (LACE) and incubated in the dark did not show a significant difference in eGFP levels compared to control groups transfected with empty plasmid. Strains containing the LACE system and gRNA exhibited significantly brighter eGFP fluorescence intensity when illuminated compared to when incubated in the dark.Activation of the eGFP reporter in strains transfected with the gRNA and LACE constructs, the gRNA and tCas9-VP64 expression plasmid or an empty plasmid as a negative control was quantified after 24 hours of illumination or incubation in the dark.

Figure 6: Activation capability of LACE system using gRNA-R1 plasmid and tCas9-CIBN plasmid in BL21.
Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours using 15mM L-arabinose at 30°C.

Proof of expression

Stationary cultures of BL21 J23114 was subcultured into fresh media and growth for 4 hours. Subsequent Extraction of protein from Bacterial and visualization using SDS-PAGE confirms that proteins of the expected size are present in the supernatant and hence most likely successfully secreted by the engineered bacterial strains.

Figure 7: Western blot analysis of CRY2-VP64 protein levels.
1:Control groups transfected with empty plasmid; 2 3 4:transfected with CRY2-VP64 plasmid (J23114 promoter) from different single colonies, ~78.5 kDa. Stationary cultures of BL21 was subcultured into fresh media and growth for 4 hours at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 405
Illegal BglII site found at 864
Illegal BamHI site found at 1343
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 289
Illegal AgeI site found at 1018
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 641
Illegal BsaI.rc site found at 50
Illegal SapI.rc site found at 158

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td.AutoAnnotatorGO1 { width: 5% } td.AutoAnnotatorGO2 { width: 35% } td.AutoAnnotatorGO3 { width: 60% } td.AutoAnnotatorPredFeat1 { width:3% } td.AutoAnnotatorPredFeat2a { width:27% } td.AutoAnnotatorPredFeat3 { width:70% } div.AutoAnnotator_trans { position:absolute; background:rgb(11,140,143); background-color:rgba(11,140,143, 0.8); height:5px; top:100px; } div.AutoAnnotator_sec_helix { position:absolute; background:rgb(102,0,102); background-color:rgba(102,0,102, 0.8); height:5px; top:110px; } div.AutoAnnotator_sec_strand { position:absolute; background:rgb(245,170,26); background-color:rgba(245,170,26, 1); height:5px; top:110px; } div.AutoAnnotator_acc_buried { position:absolute; background:rgb(89,168,15); background-color:rgba(89,168,15, 0.8); height:5px; top:120px; } div.AutoAnnotator_acc_exposed { position:absolute; background:rgb(0, 0, 255); background-color:rgba(0, 0, 255, 0.8); height:5px; top:120px; } div.AutoAnnotator_dis { position:absolute; text-align:center; 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_1473655335837'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1473655335837 --><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;<u>ATGAAGATG&nbsp;...&nbsp;GGTTGCAAA</u>TAATAA</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 1 to 1836 (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;<br>401&nbsp;<br>501&nbsp;<br>601&nbsp;</td><td class="AutoAnnotatorSeqSeq">MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVF<br>NHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEK<br>PSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNCTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYI<br>CFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYI<br>SGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPD<br>EIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSD<br>QITTSLGKNGCK*</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="AutoAnnotatorSeqFeat2b">None of the supported features appeared in the sequence</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">47 (7.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">33 (5.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">21 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">35 (5.7%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">12 (2.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">18 (2.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">48 (7.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">36 (5.9%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">13 (2.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">36 (5.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">59 (9.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">38 (6.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">12 (2.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">25 (4.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">31 (5.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">54 (8.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">24 (3.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">22 (3.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">17 (2.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">31 (5.1%)</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">612</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">71 (11.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">83 (13.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">77 (12.6%)</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>3125</sub>H<sub>4814</sub>N<sub>836</sub>O<sub>913</sub>S<sub>24</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">69473.1</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.69</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">146330 / 147080 (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_1473655335837()' 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 id="hydrophobicity_charge_placeholder2" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder3" 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 (0.69)</td><td class="AutoAnnotatorCodonUsage3">good (0.73)</td><td class="AutoAnnotatorCodonUsage3">good (0.69)</td><td class="AutoAnnotatorCodonUsage3">good (0.78)</td><td class="AutoAnnotatorCodonUsage3">good (0.80)</td><td class="AutoAnnotatorCodonUsage3">good (0.67)</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_1473655335837(){hydrophobicity_datapoints = 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= undefined;trans_datapoints = undefined;sec_helix_datapoints = undefined;sec_strand_datapoints = undefined;acc_exposed_datapoints = undefined;acc_buried_datapoints = undefined;flot_plot_options = []; flot_plot_options[0] = {grid: {borderWidth: {top: 0,right: 0,bottom: 0,left: 0}},legend: {show: false},xaxes: [{show: true,min: 0,max: 200,ticks: [[0.5, '1'], [24.5, '25'], [49.5, '50'], [74.5, '75'], [99.5, '100'], [124.5, '125'], [149.5, '150'], [174.5, '175'], [199.5, '200']],tickLength: -5}],yaxes: [{show: true,ticks: [[0, '0'], [4.5,'hydro-<br>phobic&nbsp;&nbsp;'], [-4.5,'hydro-<br>philic&nbsp;&nbsp;']],min: -4.5,max: +4.5,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(100,149,237,1)'}},{show: true,ticks: [[0, ''], [1,'positive<br>&nbsp;charge'], [-1,'negative<br>&nbsp;charge']],position: 'right',min: -1,max: 1,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(255,99,71,1)'}}]};number_of_plots = 4;for ( plot_num = 1 ; plot_num < number_of_plots ; plot_num ++){flot_plot_options[plot_num] = jqAutoAnnotator.extend(true, {} ,flot_plot_options[0]);flot_plot_options[plot_num].xaxes = [{min: plot_num*200,max: (plot_num + 1)*200,ticks: [ [plot_num*200 +   0.5, (plot_num*200 +   1).toString()], [plot_num*200 +  24.5, (plot_num*200 +  25).toString()], [plot_num*200 +  49.5, (plot_num*200 +  50).toString()], [plot_num*200 +  74.5, (plot_num*200 +  75).toString()], [plot_num*200 +  99.5, (plot_num*200 + 100).toString()], [plot_num*200 + 124.5, (plot_num*200 + 125).toString()], [plot_num*200 + 149.5, (plot_num*200 + 150).toString()], [plot_num*200 + 174.5, (plot_num*200 + 175).toString()], [plot_num*200 + 199.5, (plot_num*200 + 200).toString()] ],tickLength: -5}];};try {if( jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_button').val() =='Show' 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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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#hydrophobicity_charge_placeholder'+ plot_num.toString(), plot_datasets, flot_plot_options[plot_num] );}var screen_width = jqAutoAnnotator('canvas.flot-base').width(); var pos_of_first_tick = 46;var pos_of_last_tick = screen_width - 51;var tick_diff = (screen_width - 97)/199;if(jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #dis_checkbox').prop('checked') == true){for ( j = 0 ; j < dis_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][0] - 1)*tick_diff - Math.floor((dis_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][1] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][1] - 1)*tick_diff - Math.floor((dis_datapoints[j][1] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #trans_checkbox').prop('checked') == true){for ( j = 0 ; j < trans_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_placeholder' + Math.floor((trans_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_trans\' style=\'width:' + (((trans_datapoints[j][1] - trans_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px ;left:' + ((pos_of_first_tick + (trans_datapoints[j][0] - 1.5)*tick_diff - Math.floor((trans_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #sec_checkbox').prop('checked') == true){for ( j = 0 ; j < sec_helix_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_placeholder' + Math.floor((sec_helix_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_helix\' style=\'width:' + (((sec_helix_datapoints[j][1] - sec_helix_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_helix_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_helix_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}for ( j = 0 ; j < sec_strand_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1473655335837 #hydrophobicity_charge_placeholder' + Math.floor((sec_strand_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_strand\' style=\'width:' + (((sec_strand_datapoints[j][1] - sec_strand_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_strand_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_strand_datapoints[j][0] - 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-<!-- -->
+==Proof of function==
-<span class='h3bb'>Sequence and Features</span>
+''' Verification of the suppression efficiency of gRNA'''<br>
+With [https://parts.igem.org/part:BBa_K1982006 (BBa_K1982006)] different target loci have been tested by the usage of a GFP reporter plasmid(pCold-1) with a CSPA promotor. The target sites can be determined by directing the gRNA consisting of 20 bp length against the desired sequence of interest. R1 with target sites at different distances to the promotor regions proved successfully as potential activation sites (see Table 1 and Figure 3).
+   <table class="table table-striped table-bordered table-responsive text-center">
+                                <tr>
+                                    <td><b>Name</b></td>
+                                    <td><b>Binding Site</b></td>
+                                    <td><b>Distance to promoter</b></td>
+                                    <td><b>Sequence</b></td>
+                                    <td><b>Position</b></td>
+                                </tr>
+                                <tr>
+                                    <td>F1</td>
+                                    <td>CSPA promoter</td>
+                                    <td>15</td>
+                                    <td>TGCATCACCCGCCAATGCG</td>
+                                    <td>sense sequences</td>
+                                </tr>
+                                <tr>
+                                    <td>F2</td>
+                                    <td>non-coding</td>
+                                    <td>68</td>
+                                    <td>GCCGCCGCAAGGAATGGTG</td>
+                                    <td>sense sequences</td>
+                                </tr>
+                                <tr>
+                                    <td>R1</td>
+                                    <td>CSPA promoter</td>
+                                    <td>43</td>
+                                    <td>ATTAATCATAAATATGAAA</td>
+                                    <td>antisense sequences</td>
+                                </tr>
+                                <tr>
+                                    <td>R2</td>
+                                    <td>non-coding</td>
+                                    <td>94</td>
+                                    <td>CATCATCCAACTCCGGCAAC</td>
+                                    <td>antisense sequences</td>
+                                </tr>
+                            </table>
+                            <p CLASS="text-center">
+'''Table 1:''' Overview of the tested gRNAs with different binding sites on the GFP pCold-1 plasmid.
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/a/a9/T--NEU-China--Design4.png" width="700px"></html>
+|-
+|'''{{{title|Figure 3: Position of the target loci on the GFP pCold-1 plasmid.}}}'''
+|}
+</div>
+To ensure the suppression efficiency of the gRNA, four gRNA sequences targeting different sites of CSPA promoter were designed and transfected into the <i>E. coli</i> strains <i>BL21</i>. Efficient suppression of CSPA promoter in strains  <i>BL21</i> was observed. GFP levels in GFP transgenic strains decrease after inserting a fragment that expresses CSPA promoter gRNA. And the sequence with the best suppression effect was selected for further study.
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/b/b2/T--NEU-China--Results51.jpg" width="60%"></html>
+|}
+</div>
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/8/85/T--NEU-China--Results52.jpg" width="45%"></html>
+|-
+|'''{{{title|Figure 4: Results of the GFP-influence under the CSPA promotor<br/>only using different gRNAs targeted to CSPA promoter in <i>BL21</i>..}}}'''<br />{{{subtitle| 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter)  9:Control group transfected with GFP plasmid,~27 kDa. Excitation wavelength: 488 nm, Emission wavelength:  509nm. Stationary cultures of <i>BL21 </i>was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8) at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).}}}
+|}
+</div>
+'''Silencing capability validation'''
+We next evaluated the effect of tCas9-cibn on suppressing CSPA promoter. GFP expression levels were assayed in strains <i>BL21</i> after co-transformation with tCas9-cibn and CRY2-VP64 and gRNA in the absence of light conditions.
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/6/67/T--NEU-China--Results61.jpg" width="60%"></html>
+|}
+</div>
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/0/0e/T--NEU-China--Results62.png" width="60%"></html>
+|-
+|'''{{{title|Figure 5: Silencing capability of tCas9-CIBN with gRNA <br/> using different gRNAs targeted to CSPA promoter in <i>BL21</i>.}}}'''<br />{{{subtitle| 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) and tCas9-CIBN plasmid(pBAD promoter)  <br>9 10:Control groups transfected with GFP plasmid and  tCas9-CIBN plasmid(pBAD promoter), ~27 kDa. Excitation wavelength: 488 nm, Emission wavelength:  509nm. Stationary cultures of <i>BL21 </i>was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8 10) using 15mM L-arabinose at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).}}}
+|}
+</div>
+Compared with control groups, green fluorescence intensity and mRNA levels were dramatically reduced in groups treated with gRNA and tCas9-cibn. These results suggest that gRNA can specifically guide tCas9 to target upstream of CSPA promoter, thereby to inhibit CSPA promoter to reduce GFP expression levels.<br>
+=Activation of a fluorescence reporter=
+Spatially controlled activation of gene expression was achieved in strains co-transfected with the LACE system, a reporter vector containing a gRNA target sequence upstream of CSPA promoter and the eGFP gene. Strains transfected with light-activated CRISPR/Cas9 effector (LACE) and incubated in the dark did not show a significant difference in eGFP levels compared to control groups transfected with empty plasmid. Strains containing the LACE system and gRNA exhibited significantly brighter eGFP fluorescence intensity when illuminated compared to when incubated in the dark.Activation of the eGFP reporter in strains transfected with the gRNA and LACE constructs, the gRNA and tCas9-VP64 expression plasmid or an empty plasmid as a negative control was quantified after 24 hours of illumination or incubation in the dark.
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/4/46/T--NEU-China--Results71.jpg" width="55%"></html>
+|-
+|'''{{{title|Figure 6: Activation capability of LACE system using gRNA-R1 plasmid and tCas9-CIBN plasmid in <i>BL21</i>.}}}'''<br />{{{subtitle| Excitation wavelength: 488 nm, Emission wavelength:  509nm. Stationary cultures of <i>BL21 </i>was subcultured into fresh media and growth for 8 hours using 15mM L-arabinose at 30°C.}}}
+|}
+</div>
+==Proof of expression==
+Stationary cultures of <i>BL21</i> J23114 was subcultured into fresh media and growth for 4 hours. Subsequent Extraction of protein from Bacterial and visualization using SDS-PAGE confirms that proteins of the expected size are present in the supernatant and hence most likely successfully secreted by the engineered bacterial strains.
+<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 0px; border:{{{border|0px solid #aaa}}};width:{{{width|800px}}};padding:10px 10px 0px 0px;">
+{|
+|<html> <img src="https://static.igem.org/mediawiki/2016/5/56/T--NEU-China--Results2.png" width="90%">
+</html>
+|-
+|'''{{{title|Figure 7: Western blot analysis of CRY2-VP64 protein levels.}}}'''<br />{{{subtitle| 1:Control groups transfected with empty plasmid;      2 3 4:transfected with CRY2-VP64 plasmid (J23114 promoter) from different single colonies, ~78.5 kDa. Stationary cultures of <i>BL21 </i>was subcultured into fresh media and growth for 4 hours at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).}}}
+|}
+</div>
+<span class='h3bb'>
+==Sequence and Features==
+</span>
 <partinfo>BBa_K1982009 SequenceAndFeatures</partinfo>

Difference between revisions of "Part:BBa K1982009"

Revision as of 13:22, 15 October 2016

Proof of function

Activation of a fluorescence reporter

Proof of expression

Sequence and Features