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  1. Over expression of Acetyl- CoA carboxylase (ACC) sub-unit accCin E.coli to enhance fatty acid (triacyl glycerol) accumulation for Bio-fuel production” “Fuel it up” Teammembers: ParulSirohi SanjuTimilsina

  2. Goal: To overexpressed accC gene in E.coli to increases Tri acyl glycerol (TAG) production and then our future goal will be to express the accC gene in any possible microorganisms ( Algae and bacteria) which might enhance lipid production in waste biomass.

  3. Introduction Biochemical pathway • The gene of interest in this project is accC ( Acetyl Co-A carboxylase biotin carboxylase).This gene catalyze the formation of malonyl-CoAsubstrate for biosynthesis of fatty acid synthesis. • ACC is a multi subunit( accA, accB, accC and accD) enzyme in most prokaryotes. It is also found in the chloroplast of most of plant and algae. • Fatty acid is the major prerequisite for bio-fuel production, so its over production might enhance bio-fuel production. • Overexpression of the enzyme DGAT is most likely to enhance lipid, Tri-acyl glycerol over production but due to high introns numbers in the source ( Arabidopsis thaliana) we chose ACC over DGAT. Source: NMD courchesne et.al./ journal of biotechnology 141(2009)31-41

  4. Brief overview Source Organism: E. coli 0157:H7 Source: Biology department of University of Northern Iowa Media: Luria Broth Gene: Acetyl CoA carboxylase biotin carboxylase (accC) Assembly #: NC_011353.1 Region: 4242644..4243993 bp:1350 Introns: none ( prokaryote) Bio-brick Compatibility: Compatible

  5. PCR Primer Sequence for accC gene Primers: • 24F_Biofuel1P 5’gaattcgcggccgcttctagagatgctggataaaattgttattgccaaccgc 3’ • 24RP_Biofuel2S 5’tactagtagcggccgctgcagcgagttttttctccagatagtggatgttagtgc3’ • 24F_Biofuel1 5’ atgctggataaaattgttattgccaaccgc 3’ • 24RP_Biofuel2 5’ cgagttttttctccagatagtggatgttagtgc3’

  6. Gene sequence with primers: Forward primer: • 5’gaattcgcggccgcttctagagatgctggataaaattgttattgccaaccgc 3’      1 atgctggataaaattgttattgccaaccgcggcgagattgcattgcgtattcttcgtgcc      61 tgtaaagaactgggcatcaagactgtcgctgtgcactccagcgcggatcgcgatctaaaa     121 cacgtattactggcagatgaaacggtctgtattggccctgctccgtcagtaaaaagttat     181 ctgaacatcccggcaatcatcagcgccgctgaaatcaccggcgcagtagcaatccatccg     241 ggttacggcttcctctccgagaacgccaactttgccgagcaggttgaacgctccggcttt     301 atcttcattggcccgaaagcagaaaccattcgcctgatgggcgacaaagtatccgcaatc     361 gcggcgatgaaaaaagcgggcgtcccttgcgtaccgggttctgacggcccgctgggcgac     421 gatatggataaaaaccgtgccattgctaaacgcattggttatccggtgattatcaaagcc     481 tccggcggcggcggtggtcgcggtatgcgcgtagtgcgcggcgacgctgaactggcacaa     541 tccatctccatgacccgtgcggaagcgaaagctgctttcagcaacgatatggtttacatg     601 gagaaatacctggaaaatcctcgccacgtcgagattcaggtactggctgacggtcagggc     661 aactctatctatctggcggaacgtgactgctccatgcagcgccgccaccagaaagtggtc     721 gaagaagcaccagcaccgggcattaccccggaactgcgtcgctacatcggcgaacgttgc     781 gctaaagcgtgtgttgatatcggctatcgcggtgcaggtactttcgagttcctgttcgaa     841 aacggcgagttctatttcatcgaaatgaacacccgtattcaggtagaacacccggttaca     901 gaaatgatcaccggcgttgacctgatcaaagaacagctgcgtatcgctgccggtcaaccg     961 ctgtcgatcaagcaagaagaagttcacgttcgcggccatgcggtggaatgtcgtatcaac    1021 gccgaagatccgaacaccttcctgccaagtccgggcaaaatcacccgtttccacgcacct    1081 ggcggttttggcgtacgttgggagtctcatatctacgcgggctacaccgtaccgccgtac    1141 tatgactcaatgatcggtaagctgatttgctacggtgaaaaccgtgacgtggcgattgcc    1201 cgcatgaagaatgcgctgcaggagctgatcatcgacggtatcaaaaccaacgttgatctg    1261 cagatccgcatcatgaatgacgagaacttccagcatggtggcactaacatccactatctggagaaaaaactcggtcttcaggaaaaataa 3’cgtgattgtaggtgatagacctcttttttgagcgacgtcgccggcgatgatcat 5’ Reverse Primer

  7. Vector Plasmid pSB1A3 pSB1A3 is a high copy number plasmid carrying Ampicillin resistance pSB1A3 is a bio-brick compatible plasmid that has prefix(E-X) and suffix(S-P) once in the whole sequence Its is also compatible for the constitutive promoter family member

  8. Promoter/Regulator • Part: BBa_J23100 • Constitutive promoter family member is the consensus promoter sequence and is the strongest member of the family • RFP(au) of the J23100 is 2547 fold more than in wild type, which means it has 2547 fold more ability to increase enzymatic activity of enzyme in the biochemical pathway • Sequence: ttgacggctagctcagtcctaggtacagtgctagc • Alternative Promoters: • J23109:RFP-106 tttacagctagctcagtcctagggactgtgctagc( medium promoter) • BBa-K206000(PBad): is strong E.coli promoter controlled by L-arabinose inducer and is repressed by AraC.

  9. Steps • Grow the source organism (E. coli) in LB media • DNA extraction from the source (E. coli) • Electrophoresis to check desired DNA segment (bp) • Primer designing • Multiplication of gene of interest by PCR • Electrophorosis • Digestion of Plasmid by restriction enzymes • Ligation of accC gene in plasmid vector (pSB1A3) • Transformation of vector plasmid into host organism E. coli • Cloning of cells in a LB media • Selection for recombinant DNA colonies by antibiotic selective media (LB+ ampicillin) • Inoculation of E.coli in biomass • Testing of protein by SDS-PAGE and fatty acid (tri acyl glycerol) by thin layer chromatography -Materials for TLC: Silica gel, -Solvent mixture hexane/diethyl ether/acetic acid(17/3/0.2/v/v/v) -CuSO4 reagent ( 20g CuSO4, 200ml methanol, 8ml H2SO4 and 8ml H3PO4)) -acetone/ toluene/ water( 91/30/8,v/v/v)

  10. References • Magnuson, K., Jackowski, S., Rock, C.O., and Cronan, J.E.(1993).Regulation of fatty acid biosynthesis in Escherichia coli. Microbial Rev.57(3):522 • Noemie, M. D., Parisien, A., Wang, B., Lan, C., ( 2009). Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. Journal of biotechnology, 141 (2009) 31-41 • Siaut, M., Cuine, S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Bryly, A., Beisson, F., Triantaphylides, C., Beisson, L., and Peltier, G., (2011). Oil Accumulation in the model green algae Chlamydomonasreinhardetii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 2011: 117 • http://partsregistry.org/Main_Page • http://www.ncbi.nlm.nih.gov/ • http://scholar.google.com/ • www.wikipedia.org/