BBa_K929301 1 BBa_K929301 Potsdam Standard Backbone 2012-09-23T11:00:00Z 2015-05-08T01:13:47Z BBa_J04450 Released HQ 2013 The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method ([http://www.ncbi.nlm.nih.gov/pubmed/20646988 Blanusa ''et al.'' (2010)]). For this standard we designed a new cloning backbone with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3, the Potsdam Standard Backbone. In the prefix, we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3??? overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that???s the only step where we have to use restriction enzymes.<br> The gene of interest was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5??? end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3??? overhang which is suitable to the overhangs that was created by cutting the new assembly vector. After that the digested vector and the PLICed insert were mixed and transformed into <i>E. coli</i>. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.<br><br> false true _1194_ 0 14525 9 In stock true To create the Potsdam Standard Backbone, we amplified the RFP expression cassette (BBa_J04450) to add the restriction site for Apa I and Sph I. Then, the amplified expression cassette was insert into pSB1C3 using Xba I and Pst I. false Potsdam Bioware 2012 iGEM annotation2203447 1 Cap range2203447 1 7 44 annotation2203450 1 RFP range2203450 1 145 822 annotation2203448 1 LacI range2203448 1 83 118 annotation2203449 1 BBa B0034 range2203449 1 127 138 annotation2203446 1 Apa I range2203446 1 1 6 annotation2203451 1 Sph I range2203451 1 823 828 BBa_K929301_sequence 1 gggcccgcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaagcatgc 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