BBa_K316017 1 BBa_K316017 LacI operator controlled TEV protease 2010-10-24T11:00:00Z 2015-05-08T01:11:56Z The part was produced by nucleotide synthesis by mwg ??? eurofins. The synthetic RBS was designed to work with <bbpart>BBa_K316000</bbpart>. TEV protease S219P autocatalysis resistant variant <bbpart>BBa_K316012</bbpart>. This part had been reversed for the 3' strand in order to reduce any read-through that may be caused by upstream elements. Expression of TEV protease is under control of enhanced hyperspank promoter <bbpart>BBa_K316000</bbpart>. false false _440_ 0 7480 9 It's complicated false TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins [http://www.eurofinsdna.com/home.html] proprietary software. false IC 2010 Team component2218950 1 BBa_K316000 component2218945 1 BBa_K316012 annotation2218945 1 BBa_K316012 range2218945 1 1 717 annotation2218950 1 BBa_K316000 range2218950 1 726 874 BBa_K316012 1 TEV TEV protease S219P autocatalysis resistant variant 2010-10-22T11:00:00Z 2015-05-08T01:11:56Z TEV protease is naturally found in Tobacco Etch Virus genome. This part contains the sequence codon optimized for expression in B.subtilis using mwg ??? eurofins [[www.eurofinsdna.com]] proprietary software. TEV protease S219P autocatalysis resistant variant Introduction : This is the nuclear inclusion protease, endogenous to Tobacco Etch Virus and is used in the late lifecycle to cleave polyprotein precursors. The recognition sequence is ENLYFQG/S [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a]] between QG or QSDue to it???s stringent sequence specificity, TEV is commonly used to cleave genetically engineered proteins. Uses: TEV proteinase is used to cleave fusion proteins. It is useful due to its high degree of specificity [[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-45PMGK3-9P&_user=217827&_coverDate=02%2F01%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=e075aad3a349720ad9484095d01a65be&searchtype=a]] and potential to be used in vivo or in vitro applications. Auto-inactivation Wild type TEV protease also cleaves itself at Met 218 and Ser 219 [[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WXR-45R86R6-5X&_user=7635175&_coverDate=06%2F20%2F1995&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=7635175&md5=0a1521e783c0bd4126b145f3f6d766d4&searchtype=a]] . This leads to auto-inactivation of the TEV protease and progressive loss of activity of the protein. The rate of inactivation is proportional to the concentration of protease [[http://peds.oxfordjournals.org/content/14/12/993.long ]] More stable Mutants have been produced by single amino acid substitutions S219V (AGC(serine) to GTG(valine) and S219P (AGC(serine) to CCG(proline) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency '''Table I.''' Kinetic parameters for wild-type and mutant TEV proteases with the peptide substrate TENLYFQSGTRR-NH2. Enzyme Km (mM) kcat (s&#8722;1) kcat/Km (mM&#8722;1 s&#8722;1) Wild-type 0.061 ?? 0.010 0.16 ?? 0.01 2.62 ?? 0.46 S219V* 0.041 ?? 0.010 0.19 ?? 0.01 4.63 ?? 1.16 S219P* 0.066 ?? 0.008 0.09 ?? 0.01 1.36 ?? 0.22 S219P* - virtually imperivious to autocatalysis S219V* - retains same activity as wild type Full article can be seen here [[http://peds.oxfordjournals.org/content/14/12/993.long]] false false _440_ 0 7480 9 Not in stock false The part was produced by nucleotide synthesis by mwg ??? eurofin false IC 2010 Team annotation2100977 1 TEV protease range2100977 1 1 717 BBa_K316000 1 sRBS Pehs Reverse strand coding Enhanced LacI-hyperspank promoter 2010-10-19T11:00:00Z 2015-05-08T01:11:56Z DNA synthesis by MWG eurofins. The sequence was codon optimized for expression in B.subtilis using mwg ??? eurofins www.eurofinsdna.com proprietary software. This part is a modified version of hyper-spank promoter for B.subtilis <bbpart>BBa_K143015</bbpart>. Hyper-spank promoter is repressed by transcriptional repressor LacI <bbpart>BBa_K143033</bbpart> and can be induced by addition of Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG). Constitutive expression of LacI is required for repression. Promoter Design The position and sequence of LacI binding was designed using existing knowledge. The stochastic nature of transcriptional repressors usually leads to background transcription. In order to minimise background the binding sites and the distance between them have been optimised. Stronger binding The natural LacI operator has 3 binding sites, all of which have variations in the binding sequences. Perfectly symmetric binding sequence was shown to have10-fold higher binding compared to wild type sequences. The aattgtgagc gctcacaatt sequence has been shown to be optimal for LacI binding Muller 1996 Oehler 1994. Optimal distance Due to the tetrameric nature of LacI it can simulataneously bind to multiple regions in the genome. Binding at multple sites can produce much stronger repression (muller 1996) by increasing local LacI concentrations. Due to the helical nature of DNA the distance between the operator sites plays an important role in the strength of repression. Maximal repression at 70.5bp, second strongest at 92.5bp and third at 115.5bp false false _440_ 0 7480 9 It's complicated false Designed for minimal basal transcription by altering the LacI binding site sequence and distance between them. false IC 2010 Team annotation2097817 1 scar range2097817 1 13 20 annotation2100978 1 LacI binding range2100978 1 21 51 annotation2100979 1 LacI binding range2100979 1 111 140 annotation2097815 1 sRBS range2097815 1 1 12 BBa_K316000_sequence 1 ttcacctcctttctctagtatgtgaattgttatccgctcacaattccacacacacattatgccacaccttgtagataaagtcaacaacttttgcaactttctcggcaaaatgtggaattgtgagcgctcacaattccacaaccctcgag BBa_K316012_sequence 1 ttattagttcattaactgtgtggcctctttaaccggttgaaacggttcttccggtttatccatgaacactttgtgacctccccaaagaacggaatctgcattcagtctccagcctgaaacccattgttgagcttcttggttcgtcagtaactccatgaagttcttgggaacactcgtaaagtaattattcgtattggtgaagtttgacgcagagtgtatgcccacaataaacccatcccgtgttgacacaagaggagagccacattgcccatcttttgtctgaatccaatgcttccaaaagatgccatcagagctagggaatgtacagctagtgtctgataccattgaggacatagatttggtctgaaagtttgttgtgactaagcagatccgttcttcacgctgaggttcgcgaaacttgagtttctgaggaaacggtggaaagtctttcggcattcgaatgataatcatgtctcttccatctatcagatgttgctgaagcgtcgttgtgtttttgaccttaaagactccatgaagggattgtaccaacagtgtaccattattgcgcctaaagaggtgtttgttcgtgatgatgaatggcccaaagccaatgccatacaagctagtggtatgaccatcactctcattggtcagatggcaaatagtcgagctaatcggattatagtcacgtggtcctttaaacaacgatcttcccat BBa_K316017_sequence 1 ttattagttcattaactgtgtggcctctttaaccggttgaaacggttcttccggtttatccatgaacactttgtgacctccccaaagaacggaatctgcattcagtctccagcctgaaacccattgttgagcttcttggttcgtcagtaactccatgaagttcttgggaacactcgtaaagtaattattcgtattggtgaagtttgacgcagagtgtatgcccacaataaacccatcccgtgttgacacaagaggagagccacattgcccatcttttgtctgaatccaatgcttccaaaagatgccatcagagctagggaatgtacagctagtgtctgataccattgaggacatagatttggtctgaaagtttgttgtgactaagcagatccgttcttcacgctgaggttcgcgaaacttgagtttctgaggaaacggtggaaagtctttcggcattcgaatgataatcatgtctcttccatctatcagatgttgctgaagcgtcgttgtgtttttgaccttaaagactccatgaagggattgtaccaacagtgtaccattattgcgcctaaagaggtgtttgttcgtgatgatgaatggcccaaagccaatgccatacaagctagtggtatgaccatcactctcattggtcagatggcaaatagtcgagctaatcggattatagtcacgtggtcctttaaacaacgatcttcccattactagagttcacctcctttctctagtatgtgaattgttatccgctcacaattccacacacacattatgccacaccttgtagataaagtcaacaacttttgcaactttctcggcaaaatgtggaattgtgagcgctcacaattccacaaccctcgag igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z