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Over expression of Acetyl- CoA carboxylase (ACC ) sub-unit accC in E.coli to enhance fatty acid ( triacyl glycerol) accumulation for Bio-fuel production”. “Fuel it up” Team members: Parul Sirohi Sanju Timilsina. Goal:.

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fuel it up team members parul sirohi sanju timilsina
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

slide2
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.

introduction biochemical pathway
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

brief overview
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

pcr p rimer sequence for accc gene
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’
gene sequence with primers
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
vector plasmid psb1a3
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

promoter regulator
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.
slide9
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)

references
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/
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