Difference between revisions of "Part:BBa K2842680"
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|'''Function''' | |'''Function''' | ||
| − | | | + | |Create intein-spliced polymers |
|- | |- | ||
|'''Use in''' | |'''Use in''' | ||
| Line 42: | Line 42: | ||
| − | === | + | ===Usage and Biology=== |
| − | ==== | + | ====Protein Polymerisation by Split Inteins==== |
<p> | <p> | ||
| − | Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms | + | Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms [1]. Inteins contain both an N- and C-terminal domain, which can be split to allow either half to be bound to unique external proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction as depicted in Figure 1, which allows for the ligation of the external proteins through a peptide bond. |
</p> | </p> | ||
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</p>''' ]] | </p>''' ]] | ||
| − | Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids | + | Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids [2]. |
| − | [[File:T--UCL--bsaI digests IP RFP GFP.png|400px|thumb| | + | |
| − | <center>'''Figure | + | ====Reporter Protein==== |
| + | <p> | ||
| + | Intein Monomer 1 encodes the reporter protein, red fluorescent protein (RFP). RFPs derive from various coral species, and are utilised to emit orange and fluorescence under UV-light [3]. In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral). | ||
| + | </p> | ||
| + | |||
| + | |||
| + | |||
| + | ===Related Entries in the Registry=== | ||
| + | <p> | ||
| + | The 2018 UCL team utilised the previously existing T7 promoter (BBa_J64997), Strep-tag (BBa_K823038), and terminator (BBa_B1006) within the Intein Monomer 1 design. This part works together with Intein Monomer 2 (BBa_K2842690), which was submitted by the 2018 UCL iGEM team. | ||
| + | </p> | ||
| + | ====New Part Status==== | ||
| + | <p> | ||
| + | Other than these specified registered basic parts, the rest of our Intein Monomer 1 construct is made up of newly submitted basic parts by the 2018 UCL iGEM team, therefore potentially making Intein Monomer 1 a novel biobrick. | ||
| + | |||
| + | Intein Monomer 1 is also the only Biobrick registered with a AceL-TerL-C intein. | ||
| + | </p> | ||
| + | |||
| + | |||
| + | ===Usage=== | ||
| + | ====Polymerisation==== | ||
| + | <p> | ||
| + | Purified Intein Monomer 1 can react with Purified Intein Monomer 2 to form long polymers because of the orthogonal inteins that are complementary to each other. This polymerisation reaction occurs optimally at the combined reaction temperatures of both split intein species, which are 8°C for AceL-TerL intein and 37°C for Npu intein (Thiel et al., 2014) [4]. | ||
| + | </p> | ||
| + | ====Functionalisation==== | ||
| + | <p> | ||
| + | Functional proteins can be Gibson Assembled into Intein Monomer 1. SapI restriction enzymes are used to remove the mRFP1, which creates GGC and GAG overhangs compatible with our Gibson overhangs 5’[GGCGTTGTTTCGCATAACTGTTATAAT]3’ and 5’[TGGAGCCACCCGCAGTTCGAAAAA]3’. | ||
| + | </p> | ||
| + | ====Primer Design==== | ||
| + | <p> | ||
| + | Forward Primer 5’[GCTTCTACAAACGCGGCTTCTT]3’ <br> | ||
| + | Reverse Primer 5’[ACGACGCCGgtTACTACATTGA]3’ <br><br> | ||
| + | |||
| + | These are the primers the 2018 UCL team used to PCR amplify and sequence the overall Intein Monomer 1 construct.<br> | ||
| + | </p> | ||
| + | <br> | ||
| + | <p> | ||
| + | Forward Primer 5’[AACGGGTTCATCGCTAGTAATTGTTATAAT]3’<br> | ||
| + | Reverse Primer 5’[AGTGGATCATGGAGCCACCCG]3’<br><br> | ||
| + | |||
| + | These are the primers the 2018 UCL team used to PCR amplify BBa_K2842681, as well as during colony PCR analysis of any inserted protein of interest. | ||
| + | </p> | ||
| + | |||
| + | |||
| + | |||
| + | [[File:T--UCL--bsaI digests IP RFP GFP.png|400px|thumb|right| | ||
| + | <center>'''Figure 2: BsaI digestions'''</center> | ||
<p> | <p> | ||
| Line 66: | Line 112: | ||
*edited to show relevant bands | *edited to show relevant bands | ||
</p>''' ]] | </p>''' ]] | ||
| + | ===Experimental Approach=== | ||
| + | ====DNA Analysis==== | ||
| + | <p> | ||
| + | To confirm the part sequence size, the 2018 UCL team ran BsaI digested samples to cut out Intein Monomer 1 out of the pSB1C3 backbone. This band is shown in Figure 2.1 at around 1200 bp. | ||
| + | </p> | ||
| + | ====Protein Analysis==== | ||
| + | <p> | ||
| + | The 2018 UCL team purified Intein Monomer 1 using a Strep-tag column. We then ran our sample on a SDS page gel against a PageRuler Plus marker, as shown in Figure 3. | ||
| + | </p> | ||
| + | [[File:T--UCL--RFP sds page gel.png|400px|thumb|left| | ||
| + | <center>'''Figure 3: SDS Page gel'''</center> | ||
| + | <p> | ||
| + | (1) Purified [https://parts.igem.org/Part:BBa_K2842680 BBa_K2842680] grown in LB media<br /> | ||
| + | (2) Purified [https://parts.igem.org/Part:BBa_K2842680 BBa_K2842680] grown in TB media <br /> | ||
| + | *edited to show relevant bands | ||
| + | </p>''' ]] | ||
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| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
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| − | |||
| + | ====Fluorescence Analysis==== | ||
| + | <p> | ||
| + | The 2018 UCL team transformed Intein Monomer 1 into One Shot™ BL21 Star™ (DE3) Chemically Competent E. coli. The cells were cultured in 50mL LB growth medium at 25°C, 30°C and 37°C degrees, until OD600 reached 0.6 when 400μM IPTG was added to induce expression of mRFP1 from the T7 promoter. Fluorescence was then measured after 17 hours of induction using the BMG FLUOstar Omega plate reader. Results are shown in Table 1. | ||
| + | </p> | ||
| + | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" | ||
| + | ! colspan="4" |Table 1. Fluorescence Measurements | ||
| + | |- | ||
| + | |'''Temperature''' | ||
| + | |25°C | ||
| + | |30°C | ||
| + | |37°C | ||
| + | |- | ||
| + | |'''Grown in LB medium''' | ||
| + | |600100 | ||
| + | |470790 | ||
| + | |271950 | ||
| + | |- | ||
| + | |'''Grown in TB medium''' | ||
| + | |241545 | ||
| + | |98385 | ||
| + | |361680 | ||
| + | |} | ||
| Line 115: | Line 192: | ||
| + | ====Large Scale Protein Production==== | ||
| + | <p> | ||
| + | To scale-up production of Intein Monomer 1, the 2018 UCL team grew up cultures in 500mL shake flasks and analysed the growth at two temperatures, 25°C and 37°C. The scale-up workflow is documented in Table 2, and the results are depicted in Figures 4 and 5. | ||
| + | </p> | ||
| + | <br> | ||
| + | {| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right" | ||
| + | ! colspan="4" |Table 2. Scale-up Workflow | ||
| + | |- | ||
| + | |'''Day 1 (evening)''' | ||
| + | |'''Day 2 (morning)''' | ||
| + | |'''Day 2 (evening)''' | ||
| + | |'''Day 3 (morning)''' | ||
| + | |- | ||
| + | | transform 1 µL plasmid DNA in 30 µL competent cells (BL21),<br> plate on antibiotics and leave in 37 °C condition overnight <br><br> autoclave LB/medium | ||
| + | | for each strain, prepare 8 x 10 ml LB starter cultures in 50 ml Falcon tubes by adding the appropriate antibiotics <br><br> 10 µL Chloramphenicol is needed for 10ml LB starter. | ||
| + | | pick several colonies from the LB agar plate using a sterile loop,<br> inoculate in the starter cultures <br><br>incubate the starter cultures overnight at 37 °C, 200 rpm <br><br> autoclave the 250 ml baffled-flasks containing 50 ml of each medium | ||
| + | | measure OD600 of overnight cultures | ||
| + | |} | ||
| + | [[File:T--UCL--growth1.png|400px|thumb|left| | ||
| + | <center>'''Figure 4'''</center> | ||
| + | ''' ]] | ||
| + | [[File:T--UCL--growth2.png|400px|thumb|right| | ||
| + | <center>'''Figure 5'''</center> | ||
| + | ''' ]] | ||
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| + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
| − | |||
| − | + | ||
| − | === | + | |
| + | |||
| + | |||
| + | |||
| + | ===Sequence and Features=== | ||
| + | <partinfo>BBa_K2842680 SequenceAndFeatures</partinfo> | ||
| + | <partinfo>BBa_K2842680 parameters</partinfo> | ||
| + | |||
| + | ====Functional Parameters==== | ||
| + | <html><!--- Please copy this table containing parameters for BBa_ at the end of the parametrs section ahead of the references. ---><style type="text/css">table#AutoAnnotator {border:1px solid black; width: 100%; border-collapse:collapse;} th#AutoAnnotatorHeader { border:1px solid black; width:100%; background-color: rgb(221, 221, 221);} td.AutoAnnotator1col { width:100%; border:1px solid black; } span.AutoAnnotatorSequence { font-family:'Courier New', Arial; } td.AutoAnnotatorSeqNum { text-align:right; width:2%; } td.AutoAnnotatorSeqSeq { width:98% } td.AutoAnnotatorSeqFeat1 { width:3% } td.AutoAnnotatorSeqFeat2a { width:27% } td.AutoAnnotatorSeqFeat2b { width:97% } td.AutoAnnotatorSeqFeat3 { width:70% } table.AutoAnnotatorNoBorder { border:0px; width:100%; border-collapse:collapse; } table.AutoAnnotatorWithBorder { border:1px solid black; width:100%; border-collapse:collapse; } td.AutoAnnotatorOuterAmino { border:0px solid black; width:20% } td.AutoAnnotatorInnerAmino { border:1px solid black; width:50% } td.AutoAnnotatorAminoCountingOuter { border:1px solid black; width:40%; } td.AutoAnnotatorBiochemParOuter { border:1px solid black; width:60%; } td.AutoAnnotatorAminoCountingInner1 { width: 7.5% } td.AutoAnnotatorAminoCountingInner2 { width:62.5% } td.AutoAnnotatorAminoCountingInner3 { width:30% } td.AutoAnnotatorBiochemParInner1 { width: 5% } td.AutoAnnotatorBiochemParInner2 { width:55% } td.AutoAnnotatorBiochemParInner3 { width:40% } td.AutoAnnotatorCodonUsage1 { width: 3% } td.AutoAnnotatorCodonUsage2 { width:14.2% } td.AutoAnnotatorCodonUsage3 { width:13.8% } td.AutoAnnotatorAlignment1 { width: 3% } td.AutoAnnotatorAlignment2 { width: 10% } td.AutoAnnotatorAlignment3 { width: 87% } td.AutoAnnotatorLocalizationOuter {border:1px solid black; width:40%} td.AutoAnnotatorGOOuter {border:1px solid black; width:60%} td.AutoAnnotatorLocalization1 { width: 7.5% } td.AutoAnnotatorLocalization2 { width: 22.5% } td.AutoAnnotatorLocalization3 { width: 70% } 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_1539807777240'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1539807777240 --><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"> GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCCAAGACAAGGAGGAGTACCA<u>ATGATCAAG ... CGCTTGGCT</u><br>TAAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA</span><br> <strong>ORF</strong> from nucleotide position 94 to 1032 (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 <br>101 <br>201 <br>301 </td><td class="AutoAnnotatorSeqSeq">MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCYNGGRASMASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPF<br>AWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGA<br>LKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGAGSSSESGS<u>WSHPQFEK</u>AEYCVYGDTMVETED<br>GKIKIEDLYKRLA*</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"><i>Strep</i>-tag II: </td><td class="AutoAnnotatorSeqFeat3">278 to 285</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">18 (5.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">13 (4.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">9 (2.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">19 (6.1%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">2 (0.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">10 (3.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">29 (9.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">32 (10.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">7 (2.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">15 (4.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">16 (5.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">30 (9.6%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">11 (3.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">13 (4.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">13 (4.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">20 (6.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">17 (5.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">4 (1.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">17 (5.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">18 (5.8%)</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">313</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">43 (13.7%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">48 (15.3%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">41 (13.1%)</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>1569</sub>H<sub>2417</sub>N<sub>419</sub>O<sub>483</sub>S<sub>13</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">35294.7</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.90</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">47330 / 47455 (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> <input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1539807777240()' 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 (0.80)</td><td class="AutoAnnotatorCodonUsage3">good (0.72)</td><td class="AutoAnnotatorCodonUsage3">good (0.67)</td><td class="AutoAnnotatorCodonUsage3">good (0.75)</td><td class="AutoAnnotatorCodonUsage3">good (0.79)</td><td class="AutoAnnotatorCodonUsage3">good (0.71)</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> 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"> 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_1539807777240(){hydrophobicity_datapoints = [[2.5,1.76],[3.5,1.24],[4.5,-0.56],[5.5,-0.56],[6.5,-1.72],[7.5,-1.32],[8.5,-1.26],[9.5,-1.14],[10.5,-1.06],[11.5,-1.50],[12.5,-1.42],[13.5,-1.60],[14.5,-1.52],[15.5,0.08],[16.5,0.70],[17.5,0.70],[18.5,0.26],[19.5,0.06],[20.5,-1.54],[21.5,-2.10],[22.5,-3.64],[23.5,-2.38],[24.5,-1.12],[25.5,0.34],[26.5,0.20],[27.5,0.20],[28.5,-0.44],[29.5,-0.24],[30.5,-0.10],[31.5,1.04],[32.5,1.58],[33.5,0.96],[34.5,0.90],[35.5,-0.26],[36.5,-1.32],[37.5,-1.24],[38.5,-0.62],[39.5,-2.02],[40.5,-1.40],[41.5,-0.86],[42.5,-0.40],[43.5,0.04],[44.5,0.78],[45.5,0.26],[46.5,-0.28],[47.5,-1.36],[48.5,-0.88],[49.5,0.18],[50.5,-0.44],[51.5,-0.44],[52.5,0.82],[53.5,0.36],[54.5,-1.44],[55.5,-0.10],[56.5,-0.18],[57.5,0.10],[58.5,-1.18],[59.5,0.10],[60.5,-1.16],[61.5,-0.46],[62.5,-1.46],[63.5,0.28],[64.5,-0.80],[65.5,-0.18],[66.5,-0.74],[67.5,-1.28],[68.5,-1.56],[69.5,-1.56],[70.5,-0.58],[71.5,-0.64],[72.5,-0.02],[73.5,-1.28],[74.5,-0.66],[75.5,-2.26],[76.5,-1.64],[77.5,-2.46],[78.5,-2.08],[79.5,-2.26],[80.5,-2.26],[81.5,-2.26],[82.5,-1.50],[83.5,-1.88],[84.5,-1.76],[85.5,-0.70],[86.5,-1.40],[87.5,-0.50],[88.5,-0.58],[89.5,0.40],[90.5,-0.10],[91.5,-0.10],[92.5,-0.94],[93.5,-0.24],[94.5,-1.40],[95.5,-0.50],[96.5,-0.04],[97.5,0.60],[98.5,1.04],[99.5,1.18],[100.5,-0.28],[101.5,0.94],[102.5,1.14],[103.5,0.62],[104.5,0.48],[105.5,0.48],[106.5,0.14],[107.5,-1.32],[108.5,-1.42],[109.5,-1.18],[110.5,-0.64],[111.5,-1.98],[112.5,-0.92],[113.5,-0.92],[114.5,0.00],[115.5,-0.62],[116.5,-0.48],[117.5,-1.16],[118.5,-0.54],[119.5,-2.08],[120.5,-0.40],[121.5,-0.08],[122.5,-0.46],[123.5,-1.08],[124.5,0.38],[125.5,-1.30],[126.5,-0.22],[127.5,0.32],[128.5,1.14],[129.5,0.06],[130.5,0.14],[131.5,-0.70],[132.5,0.02],[133.5,-1.32],[134.5,-1.18],[135.5,-1.18],[136.5,-2.00],[137.5,-1.72],[138.5,-0.56],[139.5,-1.08],[140.5,0.18],[141.5,0.38],[142.5,-1.16],[143.5,-1.62],[144.5,-1.00],[145.5,-0.72],[146.5,0.82],[147.5,1.38],[148.5,2.30],[149.5,2.24],[150.5,0.70],[151.5,-0.84],[152.5,-0.86],[153.5,-1.86],[154.5,-0.96],[155.5,-0.96],[156.5,-0.96],[157.5,-0.88],[158.5,-1.42],[159.5,-1.62],[160.5,-0.02],[161.5,0.42],[162.5,-0.28],[163.5,1.26],[164.5,-0.08],[165.5,-0.22],[166.5,-0.86],[167.5,-0.16],[168.5,-1.14],[169.5,-1.06],[170.5,-1.26],[171.5,-0.68],[172.5,-0.76],[173.5,-1.32],[174.5,-0.70],[175.5,-1.58],[176.5,-0.42],[177.5,0.12],[178.5,0.12],[179.5,-0.58],[180.5,-1.04],[181.5,-2.02],[182.5,-2.02],[183.5,-1.40],[184.5,-0.80],[185.5,-0.72],[186.5,-0.22],[187.5,-0.76],[188.5,-0.82],[189.5,-1.34],[190.5,-1.54],[191.5,-1.52],[192.5,-1.62],[193.5,-1.80],[194.5,-1.80],[195.5,-1.60],[196.5,-2.06],[197.5,-1.44],[198.5,-0.36],[199.5,-0.44],[200.5,0.18],[201.5,-0.44],[202.5,0.10],[203.5,-1.44],[204.5,-0.28],[205.5,-1.10],[206.5,0.36],[207.5,-1.32],[208.5,0.22],[209.5,-0.94],[210.5,-0.74],[211.5,-1.58],[212.5,-0.88],[213.5,-2.28],[214.5,-1.76],[215.5,-1.76],[216.5,-1.32],[217.5,-1.94],[218.5,-0.46],[219.5,-0.98],[220.5,-0.42],[221.5,-0.92],[222.5,-0.48],[223.5,-0.94],[224.5,0.20],[225.5,-0.44],[226.5,-1.08],[227.5,-1.14],[228.5,-0.68],[229.5,-1.74],[230.5,-0.20],[231.5,0.26],[232.5,0.50],[233.5,0.02],[234.5,0.46],[235.5,-1.08],[236.5,-0.90],[237.5,-1.52],[238.5,-0.98],[239.5,-1.50],[240.5,0.04],[241.5,-0.52],[242.5,1.08],[243.5,0.04],[244.5,0.66],[245.5,-0.74],[246.5,-0.74],[247.5,-2.34],[248.5,-2.90],[249.5,-3.00],[250.5,-2.50],[251.5,-0.90],[252.5,0.64],[253.5,0.64],[254.5,0.20],[255.5,0.08],[256.5,-1.52],[257.5,-3.26],[258.5,-2.20],[259.5,-2.20],[260.5,-2.02],[261.5,-2.22],[262.5,-1.96],[263.5,-2.48],[264.5,-1.92],[265.5,-1.92],[266.5,-0.66],[267.5,-0.10],[268.5,-0.10],[269.5,-0.12],[270.5,-0.20],[271.5,-1.26],[272.5,-1.34],[273.5,-1.26],[274.5,-1.26],[275.5,-1.28],[276.5,-0.74],[277.5,-1.22],[278.5,-1.46],[279.5,-2.00],[280.5,-1.26],[281.5,-1.80],[282.5,-1.94],[283.5,-1.26],[284.5,-1.26],[285.5,-2.08],[286.5,-0.88],[287.5,0.74],[288.5,0.12],[289.5,0.74],[290.5,0.30],[291.5,-0.34],[292.5,-0.80],[293.5,0.30],[294.5,-0.32],[295.5,0.24],[296.5,-0.32],[297.5,-1.40],[298.5,-2.32],[299.5,-2.40],[300.5,-1.36],[301.5,-1.44],[302.5,0.16],[303.5,-0.46],[304.5,-0.38],[305.5,-0.52],[306.5,0.00],[307.5,-1.68],[308.5,-1.88],[309.5,-0.42],[310.5,-0.82]];charge_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 '], [-4.5,'hydro-<br>philic ']],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> charge'], [-1,'negative<br> 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 = 2;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_1539807777240 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br> <input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'> 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> <input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'> 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> <input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'> 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> <input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'> 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> <input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'> 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> <input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'> Predicted solvent accessability: Exposed (<img src=\'https://static.igem.org/mediawiki/2013/1/16/TUM13_exposed_icon.png\' alt=\'blue bars\' height=\'10\'></img>) and buried (<img src=\'https://static.igem.org/mediawiki/2013/0/0b/TUM13_buried_icon.png\' alt=\'green bars\' height=\'10\'></img>) residues';description_html = description_html + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1539807777240();jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1539807777240);}else{jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_explanation').html('');}}catch(err){txt='There was an error with the button controlling the visibility of the plot.\n';txt=txt+'The originating error is:\n' + err + '\n\n';alert(txt);}};function plot_according_to_selectors_1539807777240(){try{var plot_datasets = [[],[]];if(jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #hydrophobicity_checkbox').prop('checked') == true){plot_datasets[0] = { color: 'rgba(100,149,237,1)',data: hydrophobicity_datapoints,label: 'Hydrophobicity',lines: { show: true, fill: true, fillColor: 'rgba(100,149,237,0.1)' },yaxis: 1};}if(jqAutoAnnotator('#AutoAnnotator_container_1539807777240 #charge_checkbox').prop('checked') == true){plot_datasets[1] = {color: 'rgba(255,99,71,1)',data: charge_datapoints,label: 'Charge',lines: { show: true, fill: true, fillColor: 'rgba(255,99,71,0.1)' },yaxis: 2};}for (plot_num = 0 ; plot_num < number_of_plots ; plot_num ++){jqAutoAnnotator.plot('#AutoAnnotator_container_1539807777240 #hydrophobicity_charge_placeholder'+ plot_num.toString(), plot_datasets, flot_plot_options[plot_num] );}var 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| + | |||
| + | ===References=== | ||
| + | <p> | ||
| + | [1] Protein Engineering Handbook. Chapter 10: Intein in protein engineering. Gillies AR and Wood DW. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Edited by Lutz S and Bornscheuer UT. Pages 271-294. <br> | ||
| + | [2] 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> | ||
| + | [3] Miyawaki A, Shcherbakiva DM and Verkhusha VV. (2013) Red fluorescent proteins: chromophore formation and cellular applications. Curr Opin Struct Biol, 22: 679-688. <br> | ||
| + | [4] Iwai H, Zuger S, Jin J and Tam PH. (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Letters, 580: 1853-1858. | ||
| + | </p> | ||
Latest revision as of 02:38, 18 October 2018
Intein Monomer 1: RFP reporter flanked with orthogonal inteins
| Intein Monomer 1 | |
|---|---|
| Function | Create intein-spliced polymers |
| Use in | E. coli cells |
| Chassis Tested | DH5α cells, BL21* cells |
| Abstraction Hierarchy | Composite Device |
| Related Device | BBa_K2842690 |
| RFC standard | RFC10,RFC12,RFC21,RFC23 & RFC25 compatible |
| Backbone | pSB1C3 |
| Submitted by | [http://2018.igem.org/Team:UCL UCL iGEM 2018] |
This gene encodes a novel split-intein flanked reporter device which enables the use of intein splicing for any protein of interest through SapI digestion. Intein Monomer 1 was created to work in conjunction with its complimentary composite part Intein Monomer 2 to construct a intein polymerisation system.
Contents
Usage and Biology
Protein Polymerisation by Split Inteins
Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms [1]. Inteins contain both an N- and C-terminal domain, which can be split to allow either half to be bound to unique external proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction as depicted in Figure 1, which allows for the ligation of the external proteins through a peptide bond.
Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids [2].
Reporter Protein
Intein Monomer 1 encodes the reporter protein, red fluorescent protein (RFP). RFPs derive from various coral species, and are utilised to emit orange and fluorescence under UV-light [3]. In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral).
Related Entries in the Registry
The 2018 UCL team utilised the previously existing T7 promoter (BBa_J64997), Strep-tag (BBa_K823038), and terminator (BBa_B1006) within the Intein Monomer 1 design. This part works together with Intein Monomer 2 (BBa_K2842690), which was submitted by the 2018 UCL iGEM team.
New Part Status
Other than these specified registered basic parts, the rest of our Intein Monomer 1 construct is made up of newly submitted basic parts by the 2018 UCL iGEM team, therefore potentially making Intein Monomer 1 a novel biobrick. Intein Monomer 1 is also the only Biobrick registered with a AceL-TerL-C intein.
Usage
Polymerisation
Purified Intein Monomer 1 can react with Purified Intein Monomer 2 to form long polymers because of the orthogonal inteins that are complementary to each other. This polymerisation reaction occurs optimally at the combined reaction temperatures of both split intein species, which are 8°C for AceL-TerL intein and 37°C for Npu intein (Thiel et al., 2014) [4].
Functionalisation
Functional proteins can be Gibson Assembled into Intein Monomer 1. SapI restriction enzymes are used to remove the mRFP1, which creates GGC and GAG overhangs compatible with our Gibson overhangs 5’[GGCGTTGTTTCGCATAACTGTTATAAT]3’ and 5’[TGGAGCCACCCGCAGTTCGAAAAA]3’.
Primer Design
Forward Primer 5’[GCTTCTACAAACGCGGCTTCTT]3’
Reverse Primer 5’[ACGACGCCGgtTACTACATTGA]3’
These are the primers the 2018 UCL team used to PCR amplify and sequence the overall Intein Monomer 1 construct.
Forward Primer 5’[AACGGGTTCATCGCTAGTAATTGTTATAAT]3’
Reverse Primer 5’[AGTGGATCATGGAGCCACCCG]3’
These are the primers the 2018 UCL team used to PCR amplify BBa_K2842681, as well as during colony PCR analysis of any inserted protein of interest.
(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands
Experimental Approach
DNA Analysis
To confirm the part sequence size, the 2018 UCL team ran BsaI digested samples to cut out Intein Monomer 1 out of the pSB1C3 backbone. This band is shown in Figure 2.1 at around 1200 bp.
Protein Analysis
The 2018 UCL team purified Intein Monomer 1 using a Strep-tag column. We then ran our sample on a SDS page gel against a PageRuler Plus marker, as shown in Figure 3.
(1) Purified BBa_K2842680 grown in LB media
(2) Purified BBa_K2842680 grown in TB media
*edited to show relevant bands
Fluorescence Analysis
The 2018 UCL team transformed Intein Monomer 1 into One Shot™ BL21 Star™ (DE3) Chemically Competent E. coli. The cells were cultured in 50mL LB growth medium at 25°C, 30°C and 37°C degrees, until OD600 reached 0.6 when 400μM IPTG was added to induce expression of mRFP1 from the T7 promoter. Fluorescence was then measured after 17 hours of induction using the BMG FLUOstar Omega plate reader. Results are shown in Table 1.
| Table 1. Fluorescence Measurements | |||
|---|---|---|---|
| Temperature | 25°C | 30°C | 37°C |
| Grown in LB medium | 600100 | 470790 | 271950 |
| Grown in TB medium | 241545 | 98385 | 361680 |
Large Scale Protein Production
To scale-up production of Intein Monomer 1, the 2018 UCL team grew up cultures in 500mL shake flasks and analysed the growth at two temperatures, 25°C and 37°C. The scale-up workflow is documented in Table 2, and the results are depicted in Figures 4 and 5.
| Table 2. Scale-up Workflow | |||
|---|---|---|---|
| Day 1 (evening) | Day 2 (morning) | Day 2 (evening) | Day 3 (morning) |
| transform 1 µL plasmid DNA in 30 µL competent cells (BL21), plate on antibiotics and leave in 37 °C condition overnight autoclave LB/medium |
for each strain, prepare 8 x 10 ml LB starter cultures in 50 ml Falcon tubes by adding the appropriate antibiotics 10 µL Chloramphenicol is needed for 10ml LB starter. |
pick several colonies from the LB agar plate using a sterile loop, inoculate in the starter cultures incubate the starter cultures overnight at 37 °C, 200 rpm autoclave the 250 ml baffled-flasks containing 50 ml of each medium |
measure OD600 of overnight cultures |
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1165
Illegal BsaI.rc site found at 28
Illegal SapI site found at 903
Illegal SapI.rc site found at 213
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) GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCCAAGACAAGGAGGAGTACCAATGATCAAG ... CGCTTGGCT TAAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA ORF from nucleotide position 94 to 1032 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
| ||||||||||||||||||||||||||||||||||||||||||||||
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid composition:
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid counting
| Biochemical parameters
| |||||||||||||||||||||||||||||||||||||||||||||
| Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
| ||||||||||||||||||||||||||||||||||||||||||||||
| 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] Protein Engineering Handbook. Chapter 10: Intein in protein engineering. Gillies AR and Wood DW. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Edited by Lutz S and Bornscheuer UT. Pages 271-294.
[2] 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.
[3] Miyawaki A, Shcherbakiva DM and Verkhusha VV. (2013) Red fluorescent proteins: chromophore formation and cellular applications. Curr Opin Struct Biol, 22: 679-688.
[4] Iwai H, Zuger S, Jin J and Tam PH. (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Letters, 580: 1853-1858.