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Explore the recent wetlab work on bacterially deposited circuits, including TSI agar assay, phsABC PCR amplification, CuS extracellular secretion analysis. Learn about CuS electric current and extracellular Cu binding. Discover the use of secretion signals and tags for protein binding to biotin chip, with examples like Pseudoazurin and Thioredoxin. Histidine patch thioredoxins for convenient protein purifications. Read about the secretion of recombinant proteins in E. coli. Dive into the extracellular transport of pseudoazurin in E. coli and mutant thioredoxin forms with metal chelating affinity.
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Yale2010 Toward Bacterially Deposited Circuits 7-1-2010
Recent Wetlab Work • TSI agar assay of pSB74 containing transformants • Cloning work – PCR amplification of phsABC and insertion vector containing iGEM terminator phsABC B0015 phsABC B0015 phsABC B0015
Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu H2S H2S H2S H2S Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Extracellular Bound Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu H2S H2S H2S Cu H2S Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Electric current CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS CuS
Extracellular Secretion Cu Cu Cu Cu Cu Cu CuS Cu H2S H2S CuS Cu Cu CuS H2S H2S Cu Cu Cu Cu CuS Cu Cu H2S Cu Cu Cu Cu Cu Cu Cu CuS CuS CuS CuS CuS CuS CuS CuS Secretion Signal on N-terminus and Avadin tag (or Strep tag) on C-terminus so the protein will bind to the biotin chip Biotin chip
Avidin or Strep-tag biotin
Pseudoazurin Thioredoxin J Biochem. 1994 Aug;116(2):327-34. Extracellular transport of pseudoazurin of Alcaligenes faecalis in Escherichia coli using the COOH-terminal domain of Serratia marcescens serine protease. J Biol Chem. 1996 Mar 1;271(9):5059-65. Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins.
Examples of the secretion of recombinant proteins expressed as fusions to the HlyA signal sequence *Endochitinase (Trichoderma harzianum), green fluorescent protein (Aequorea victoria), human erythropoetin, and trout growth hormone. http://wolfson.huji.ac.il/expression/local/bacteria/Recombinant%20protein%20secretion%20in%20Escherichia%20coli-2005.pdfz
