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    Presented by

    Dr. Sanjay Kumar,

    Assistant Professor

    Department of Microbiology

    M. D. University, Rohtak, Haryana

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    Proteins are growth associated

    products so we need to keep

    the cells growing for continuous

    protein production

    Theoretical maximum cell

    density for E.coliis 400 g DCW/L

    Maximum possible cell density

    in real for E.coliis 190 g to 200 g

    DCW/L .

    No growth possible beyond it

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    A large Metabolic flux diversion towards Biomass formation results in

    low product yield with respect to substrate consumed

    Growing cells undergo random Mutations which may cause loss of

    productivity

    Plasmid instability

    Limited operational time

    Few drawbacks associated with cell growth

    Quiescent cells can provide a solution but there are few

    challenges Not much useful for intracellular products/proteins

    No ideal quiescent cells are available

    No straightforward approach for making Quiescent cells

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    Preparation of Genomic Library of E.coliunder lac operon in pRSET A plasmid for

    random gene silencing

    Transformation in BL21 pLysS

    Screening of slow growing/Not growing but metabolically active cells by

    monitoring growth and glucose uptake rate in submerged culture

    Co-expression studies for examining the screened clones to induce growth

    retardation and enhance recombinant protein expression (using GFP as model

    protein in pBAD33 plasmid expression system under Ara operon.

    Primary screening gave 728 potentialclones

    Secondary screening gave 70 potential

    clones

    Screening of slow growing clones on IPTG supplemented LB agar plates

    Around 8000 transformants

    obtained

    17 clones were selected and genes

    involved were identified

    Methodology for first strategy for preparing Quiescent cells

    Best performer carrying gene ribB ((rib3,4 dihydroxy-2butanone-4-phosphatesynthase), gave 7 folds increase over control culture with 347 AU/g DCW

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    Results

    Growth profiles of

    induced &

    uninduced cultures

    of 17 selectedclones

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    ResultsFew transcripts whose blockage lead to growth stoppage

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    ResultsGrowth and product profiles of induced & uninduced

    cultures of Best performers (ribB & mfd)

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    Preparation of Genomic Library of E.coliunder lac operon in pBAD32 plasmid for

    random gene silencing

    Transformation in DH5 alpha carrying plasmid pNER31-GFP and grown on agar

    plate supplemented with Arabinose and IPTG as inducers

    Screening of slow growing colonies showing intense green fluorescence

    Around 30,000 transformants

    were screened

    Finally 4 clones were selected as best

    performers and their genes were

    identified

    Methodology for 2nd strategy

    Selected colonies were grown in submerged culture and growth and specific yield

    profiles were observed

    Best performer identified as kdpF

    showed 8.4 folds higher specific yield

    than that of control

    The results were reproduced using three different media showing that observed

    leap in expression was independent of media composition.

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    Results

    Growth profiles

    of selected

    betterperformers

    screened by

    using 2nd

    strategy

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    ResultsGrowth and product profiles of best performer (2

    octaprenyl-phenol hydroxylase enzyme) screened using 2nd

    strategy

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    Conclusion: A novel strategy for generating a library , consisting of randomly down

    regulated metabolic pathways in E. coli was designed by cloning small

    genomic DNA fragments in expression vector which transcribed antisenseRNA upon induction.

    Genes like ribB (rib3,4 dihydroxy-2butanone-4-phosphate synthase), mfd

    (mutation frequency decline protein) kdpF (2 octaprenyl-phenol

    hydroxylase enzyme) were found to be useful targets for obtainingslow/no growth and high expression of recombinant protein.

    No direct link at the pathway level between gene function and observed

    phenotype could be established. The observed phenotype could be

    pertained to complex regulatory response within the cells.

    Thus a high throughput screening approach was designed which is a

    useful tool for reverse metabolic engineering strategy for the generation

    of improved hosts. The approach does not rely on prior knowledge of the

    regulated and interconnected nature of cell metabolic network.

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    Thank you

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    Efficient and specific gene knockdown by small interfering RNAs produced in

    bacteria

    Linfeng Huang, Jingmin Jin, Padraig Deighan, Evgeny Kiner, Larry McReynolds, and

    Judy Lieberman

    Nature Biotechnology 31 (4) 350 - 356 doi:10.1038/nbt.2537

    Additional information

    Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate

    gene function in eukaryotic cells and may be used for therapeutic purposes to

    knock down genes implicated in disease. Thus far, most synthetic siRNAs have

    been produced by chemical synthesis. Here we present a method to produce

    highly potent siRNAs in Escherichia coli. This method relies on ectopic expression

    of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E.

    coli, p19 stabilizes an 21-nt siRNA-like species produced by bacterial RNase III.

    When mammalian cells are transfected by them, siRNAs that were generated in

    bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a

    target gene reproducibly knock down target gene expression by 90% without

    immunogenicity or off-target effects. Because bacterially produced siRNAs contain

    multiple sequences against a target gene, they may be especially useful forsuppressing polymorphic cellular or viral genes.