BBa_K2150013 1 tetX-GFP A fusion protein combining tetX(BBa_K2150101) with GFP(BBa_E0040) 2016-10-08T11:00:00Z 2016-10-19T01:06:12Z The source of tetX is the same as (xxx), the protein linker is provided by our advisor Li Hua, and the GFP is from BBa_E0040. We combine this three parts using overlap extension polymerase chain reaction (OE-PCR). This protein(noted as tetX-GFP) combines a tetracycline-degrading enzyme (tetX monooxygenase, ) with a green fluorescent protein (GFPmt3, BBa_E0040). TetX-GFP has activities of both the tetX and the GFP. As a degradation enzyme, it can degrade tetracycline(tc) and its analogs as we describe (here), and also give the tetracycline resistance to E.coli. As a green fluorescent protein, it inherits the normal function of BBa_E0040. However, the expression quantity of this protein may be smaller than BBa_E0040 under the same promoter or it may take longer time for tetX-GFP to mature, for we notice that when it is expressed under the same promoter as BBa_E0040, the fluorescent intensity(FI) of tetX-GFP is weaker than BBa_E0040 at the same growth stage of E.coli, and it takes longer for E.coli expressing tetX-GFP to reach the same FI as E.coli expressing BBa_E0040. We use tetX-GFP as a monitor of the quantity of tetX expressed in E.coli. TetX-GFP can also act as a reporter of whether our system is working (meaning whether our machine is degrading tetracycline). When tetX-GFP is expressed under a tetracycline-regulated promoter such as BBa_R0040, it will become a visible sensor of tetracycline in the presence of tetR, with no need for additional tetracycline-resistance genes. false false _2620_ 29891 29891 9 false The protein linker is 3*GGGGS which provides flexibility to the fusion protein. When choosing the codon for this linker, we need to avoid repeated sequence which may cause problems when assemble these parts. false Yang Ye annotation2489537 1 TetX range2489537 1 1 1171 annotation2489538 1 protein linker range2489538 1 1172 1215 annotation2489539 1 GFP range2489539 1 1216 1935 BBa_K2150026 1 BBa_K2150026 pT7+pTet+RBS+tetX-GFP(fusion protein)+RBS+T7RNAP+DT 2016-10-11T11:00:00Z 2016-10-17T08:48:53Z pT7:PCR amplified from BBa_K525998;T7RNAP:PCR amplified from genome of BL21. As for fusion protein tetX-GFP, tetX is the same as (xxx), the protein linker is provided by our advisor Li Hua, and the GFP is from BBa_E0040. We combine this three parts using overlap extension polymerase chain reaction (OE-PCR). pTet from BBa_R0040, DT from BBa_B0015,RBS from BBa_B0034 A positive feedback device to over-express the fusion protein of tetX and GFP. true false _2620_ 29891 31283 9 false PT7 is placed upstream of pTet, regarding that pT7 is a strong promoter while pTet is relatively weak. Linker for fusion protein is carefully selected to avoid repeated sequence that may cause problems in assembly. false Jianyi Huang component2495984 1 BBa_R0040 component2495994 1 BBa_K2150013 component2495990 1 BBa_B0034 component2495998 1 BBa_I2032 component2495996 1 BBa_B0034 component2496005 1 BBa_B0015 component2495983 1 BBa_K2150031 annotation2495998 1 BBa_I2032 range2495998 1 2070 4724 annotation2495990 1 BBa_B0034 range2495990 1 91 102 annotation2496005 1 BBa_B0015 range2496005 1 4733 4861 annotation2495983 1 BBa_K2150031 range2495983 1 1 20 annotation2495994 1 BBa_K2150013 range2495994 1 109 2043 annotation2495996 1 BBa_B0034 range2495996 1 2052 2063 annotation2495984 1 BBa_R0040 range2495984 1 29 82 BBa_K2150031 1 pT7 T7 promoter without RBS 2016-10-11T11:00:00Z 2016-10-12T09:06:12Z PCR amplified from BBa_K525998 This part is the T7 promoter without RBS. It has been reported that T7 promoter doesn't work with E.coli RNA polymerase, but only with T7 RNA polymerase(T7RNAP). However, we observed leakage of it in the absence of T7RNAP. false false _2620_ 31283 31283 9 false We deleted the RBS in BBa_K525998, so that RBS with different efficiency can be used. false Jianyi Huang BBa_I2032 1 BBa_I2032 T7 RNA polymerase 2007-04-11T11:00:00Z 2016-10-14T06:52:19Z Bacteriophage T7 <link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=10461> Released HQ 2013 Gene 1 coding region from bacteriophage T7 encoding a RNAP polymerase (RNAP). This single-protein RNAP recognizes a 21 bp consensus promoter sequence and is orthogonal to <i>E. coli</i> RNAP. false true _11_ 27399 135 84 In stock false n/a false Bartholomew Canton annotation1928674 1 putative E. coli promoter range1928674 1 2498 2525 BBa_B0034 1 BBa_B0034 RBS (Elowitz 1999) -- defines RBS efficiency 2003-01-31T12:00:00Z 2015-08-31T04:07:20Z Released HQ 2013 RBS based on Elowitz repressilator. false true _1_ 0 24 7 In stock false Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. <p>No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: <a href="mailto:(bchow@media.mit.edu)">Brian Chow</a> true Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. annotation23325 1 conserved range23325 1 5 8 BBa_B0015 1 BBa_B0015 double terminator (B0010-B0012) 2003-07-16T11:00:00Z 2015-08-31T04:07:20Z Released HQ 2013 Double terminator consisting of BBa_B0010 and BBa_B0012 false true _1_ 0 24 7 In stock false true Reshma Shetty component1916612 1 BBa_B0012 component1916610 1 BBa_B0010 annotation1916610 1 BBa_B0010 range1916610 1 1 80 annotation1916612 1 BBa_B0012 range1916612 1 89 129 BBa_B0010 1 BBa_B0010 T1 from E. coli rrnB 2003-11-19T12:00:00Z 2015-08-31T04:07:20Z Transcriptional terminator consisting of a 64 bp stem-loop. false false _1_ 0 24 7 In stock false true Randy Rettberg annotation7018 1 BBa_B0010 range7018 1 1 80 annotation4184 1 stem_loop range4184 1 12 55 BBa_B0012 1 BBa_B0012 TE from coliphageT7 2003-01-31T12:00:00Z 2015-08-31T04:07:20Z Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>. Released HQ 2013 Transcription terminator for the <i>E.coli</i> RNA polymerase. false false _1_ 0 24 7 In stock false <P> <P>Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG).<P> Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator. false Reshma Shetty annotation1687 1 stop range1687 1 34 34 annotation1690 1 polya range1690 1 28 41 annotation1686 1 T7 TE range1686 1 8 27 annotation7020 1 BBa_B0012 range7020 1 1 41 BBa_R0040 1 p(tetR) TetR repressible promoter 2003-01-31T12:00:00Z 2015-05-08T01:14:14Z Lutz, R., Bujard, H., <em>Nucleic Acids Research</em> (1997) 25, 1203-1210. Released HQ 2013 Sequence for pTet inverting regulator driven by the TetR protein.</P> false true _1_ 0 24 7 In stock false <P> <P>BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.<P> true June Rhee, Connie Tao, Ty Thomson, Louis Waldman annotation1986784 1 BBa_R0040 range1986784 1 1 54 annotation1986785 1 -35 range1986785 1 20 25 annotation1986783 1 TetR 1 range1986783 1 1 19 annotation1986787 1 -10 range1986787 1 43 48 annotation1986786 1 TetR 2 range1986786 1 26 44 BBa_B0010_sequence 1 ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc BBa_K2150026_sequence 1 taatacgactcactatagggtactagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaagaggagaaatactagatgaaaaaaaccatgcgtatcgacaccgacaaacagatgaacctgctgtctgacaaaaacgttgcgatcatcggtggtggtccggttggtctgaccatggcgaaactgcttcagcagaacggtatcgacgtttctgtttacgaacgtgacaacgaccgtgaagcgcgtatcttcggtggtaccctggacctgcacaaaggttctggtcaggaagcgatgaaaaaagcgggtctgcttcagacctactacgacctggcgctgccgatgggtgttaacatcgcggacaaaaaaggtaacatcctgtctaccaaaaacgttaaaccggaaaaccgtttcgacaacccggaaatcaaccgtaacgacctgcgtgcgatcctgctgaactctctggaaaacgacaccgttatctgggaccgtaaactggttatgctggaaccgggtaaaaaaaaatggaccctgaccttcgaaaacaaaccgtctgaaaccgcggacctggttatcctggcgaacggtggtatgtctaaagttcgtaaattcgttaccgacaccgaagttgaagaaaccggtaccttcaacatccaggcggacatccaccagccggaaatcaactgcccgggtttcttccagctgtgcaacggtaaccgtctgatggcgtctcaccagggtaacctgctgttcgcgaacccgaacaacaacggtgcgctgcacttcggtatctctttcaaaaccccggacgaatggaaaaaccagacccaggttgacttccagaaccgtaactctgttgttgacttcctgctgaaagagttctctgactgggacgaacgttacaaagaactgatccacaccaccctgtctttcgttggtctggcgacccgtatcttcccgctggaaaaaccgtggaaatctaaacgtccgctgccgatcaccatgatcggtgacgcggcgcacctgatgccgccgttcgcgggtcagggtgttaactctggtctggttgacgcgctgatcctgtctgacaacctggcggacggtaaattcaactctatcgaagaagcggttaaaaactacgaacagcagatgttcatgtacggtaaagaagcgcaggaagaatctacccagaacgaaatcgaaatgttcaaaccggacttcaccttccagcagctgctgaacgttggtggaggaggttctggaggcggtggaagtggtggcggaggtagcatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagaaagaggagaaatactagatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata BBa_B0034_sequence 1 aaagaggagaaa BBa_K2150031_sequence 1 taatacgactcactataggg BBa_R0040_sequence 1 tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac BBa_I2032_sequence 1 atgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaataa BBa_B0012_sequence 1 tcacactggctcaccttcgggtgggcctttctgcgtttata BBa_K2150013_sequence 1 atgaaaaaaaccatgcgtatcgacaccgacaaacagatgaacctgctgtctgacaaaaacgttgcgatcatcggtggtggtccggttggtctgaccatggcgaaactgcttcagcagaacggtatcgacgtttctgtttacgaacgtgacaacgaccgtgaagcgcgtatcttcggtggtaccctggacctgcacaaaggttctggtcaggaagcgatgaaaaaagcgggtctgcttcagacctactacgacctggcgctgccgatgggtgttaacatcgcggacaaaaaaggtaacatcctgtctaccaaaaacgttaaaccggaaaaccgtttcgacaacccggaaatcaaccgtaacgacctgcgtgcgatcctgctgaactctctggaaaacgacaccgttatctgggaccgtaaactggttatgctggaaccgggtaaaaaaaaatggaccctgaccttcgaaaacaaaccgtctgaaaccgcggacctggttatcctggcgaacggtggtatgtctaaagttcgtaaattcgttaccgacaccgaagttgaagaaaccggtaccttcaacatccaggcggacatccaccagccggaaatcaactgcccgggtttcttccagctgtgcaacggtaaccgtctgatggcgtctcaccagggtaacctgctgttcgcgaacccgaacaacaacggtgcgctgcacttcggtatctctttcaaaaccccggacgaatggaaaaaccagacccaggttgacttccagaaccgtaactctgttgttgacttcctgctgaaagagttctctgactgggacgaacgttacaaagaactgatccacaccaccctgtctttcgttggtctggcgacccgtatcttcccgctggaaaaaccgtggaaatctaaacgtccgctgccgatcaccatgatcggtgacgcggcgcacctgatgccgccgttcgcgggtcagggtgttaactctggtctggttgacgcgctgatcctgtctgacaacctggcggacggtaaattcaactctatcgaagaagcggttaaaaactacgaacagcagatgttcatgtacggtaaagaagcgcaggaagaatctacccagaacgaaatcgaaatgttcaaaccggacttcaccttccagcagctgctgaacgttggtggaggaggttctggaggcggtggaagtggtggcggaggtagcatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa BBa_B0015_sequence 1 ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z