Part:BBa_K3262002

J23115+Estrogen-SensitiveT7RNAP

Estrogen sensitive T7 RNA polymerase wit constitutive promoter

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NotI site found at 2577
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 819
  Illegal XhoI site found at 800
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2315
  Illegal AgeI site found at 1418
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1145
  Illegal BsaI.rc site found at 2606
  Illegal SapI.rc site found at 560
  

Overlap Extension PCR to develop construct (2019 SNU_India)

For our bacterial system, we planned to use the estrogen sensitive T7 RNA polymerase originally built by CMU iGEM team in 2014-15. Unfortunately, the part registry page (BBa_K1732015) showed that the estrogen sensitive T7 polymerase has an incomplete or erroneous sequence. According to information on the original part page, it displayed a total size of 2301bp, which is much smaller than the expected functional gene, given that just the gene for T7 RNA polymerase with the ER-LBD (estrogen responsive-ligand binding domain) is greater than 3kb. The sequence reads tab for the sample submission mentioned that the sequence read is good but inconsistent with part sequence. Furthermore, the listed sub-parts include J23115 promoter, RBS and BBa_K1491022, which is titled as ‘N-Terminus of T7 RNA Polymerase’, which is not the entire estrogen sensitive T7 RNA polymerase. We could not find any other part in the registry in which the ER-LBD had been fused with the wild type T7 RNAP. Hence, we attempted to re-design the amino-acid sequence of this protein as described by McLachlan et.al; 2011. We fused the ER-LBD between positions 179 and 180 aa residues of the T7 RNAP. These domains of the T7 RNAP were described by Shis DL and Benett MR, 2013. Unfortunately, the resulting sequence size exceeded the maximum gBlocks size of 2.3 kb. So, our strategy was to synthesize the whole sequence in two fragments: a 1.4kb N-terminal fragment featuring Promoter, RBS, T7RP positions 1-179 and ER-LBD, and a 2.1 kb fragment with the remaining T7RP positions 180-883. Our strategy was to assembled by Overlap-Extension Polymerase Chain Reaction. The peptide sequences are as follows: >T7_pol_Nterminal (1-179) MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYK >ER_LBD_HERA (312-595) ADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV >T7_Pol_Cterminal (179-883) KAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA >Complete protein sequence of Estrogen_Sensitive_T7_polymerase (1170AA) (~3.5kb) MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATVKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA The peptide sequence (Complete protein sequence of estrogen_Sensitive_T7_polymerase) was then submitted, translated and made gBlock synthesis compatible and codon-optimised for expression in E. coli on IDT server into two gblocks NT_ESRP and CT_ESRP containing the features as mentioned above. The synthesized gBlock fragments received were suspended in nuclease-free water and checked by agarose gel electrophoresis (Figure 1).

Figure 1:Agarose gel electrophoresis of IDT gBlocks suspended in milliQ and loaded on 1% agarose gel.

As seen in the gels the 1.4 kb NT-ESRP gBlock fragment showed band of expected size, however, 2.1 kb CT_ESRP gBlock had contaminating band at around 1.5kb along with the expected band of 2.1 kb. These fragments were then PCR-amplified with primers containing overlap overhang sequences for joining the fragments (Figure 2). However, the 2.1 kb fragment always gave unwanted amplification of a 1.5 kb fragment which also contaminated the original gBlock product.