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    Intro To Biohacking: or "How I Learned To Stop Worrying and Love The Zombie Apocalypse"

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    rasromeo
    This document provides an introduction to biohacking, covering key concepts in molecular biology, common techniques used such as PCR and cloning, and tools needed for a bio lab. It discusses the central dogma of DNA transcription and translation. Safety is important when working with reagents, centrifuges, and potentially hazardous organisms. The biohacking community aims to make techniques more accessible and affordable while prioritizing safety.

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    Intro To Biohacking: or "How I Learned To Stop Worrying and Love The Zombie Apocalypse"

    Uploaded by

    rasromeo
    426 views31 pages
    This document provides an introduction to biohacking, covering key concepts in molecular biology, common techniques used such as PCR and cloning, and tools needed for a bio lab. It discusses the central dogma of DNA transcription and translation. Safety is important when working with reagents, centrifuges, and potentially hazardous organisms. The biohacking community aims to make techniques more accessible and affordable while prioritizing safety.

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    nmn,m

    Original Title

    Intro to BioHacking

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    This document provides an introduction to biohacking, covering key concepts in molecular biology, common techniques used such as PCR and cloning, and tools needed for a bio lab. It discusses the central dogma of DNA transcription and translation. Safety is important when working with reagents, centrifuges, and potentially hazardous organisms. The biohacking community aims to make techniques more accessible and affordable while prioritizing safety.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    426 views31 pages

    Intro To Biohacking: or "How I Learned To Stop Worrying and Love The Zombie Apocalypse"

    Uploaded by

    rasromeo
    This document provides an introduction to biohacking, covering key concepts in molecular biology, common techniques used such as PCR and cloning, and tools needed for a bio lab. It discusses the central dogma of DNA transcription and translation. Safety is important when working with reagents, centrifuges, and potentially hazardous organisms. The biohacking community aims to make techniques more accessible and affordable while prioritizing safety.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 31
    At a glance
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    The key takeaways are an introduction to biohacking, the central dogma of molecular biology, and some common tools and concepts used in a bio lab.

    The main steps involved in cloning are extracting the desired gene, inserting it into a plasmid, transforming the plasmid into a host organism where it will express the gene.

    Some common tools used in a bio lab include a centrifuge, electrophoresis tank, incubator/water bath, thermal cycler, spectrophotometer and safety equipment.

    INTRO TO

    BIOHACKING
    Or How I learned to stop worrying and love
    the zomie apo!alypse"
    #tr$!t$re

    %hat is Bioha!&ing'

    Crash !o$rse in (ole!$lar Biology

    Key Con!epts and Tools )or a io la

    %hat are other ioha!&ers $p to'

    %hat prolems are the !omm$nity )a!ing*


    how !an we solve them'

    +is!$ssion , o$r pro-e!t ideas and goals


    B$t .irst/
    A dis!laimer000
    B$t .irst/
    A dis!laimer000
    (e +r Roert Neville
    #o000 Bioha!&ing* %T.'

    Two main streams o) development 1


    resear!h/

    Biologi!al resear!h 1 engineering

    Cloning

    2random3 m$tagenesis

    4hysi!al 1 !hemi!al resear!h and engineering

    B$ilding open5so$r!e* !ost5e))e!tive la e6$ipment


    Central +ogma" o) mol iol
    +NA
    RNA
    4rotein
    %or&
    Central +ogma" o) mol iol
    In)ormation is en!oded in o$r +NA
    O$r +NA !ontains )o$r ases/ A C T G
    These se6$en!es are re!ipes" )or
    man$)a!t$ring proteins* whi!h do all the wor& in
    the !ell
    H$mans have 78 illion ase pairs en!oding
    appro9 :;5:<*;;; protein5!oding genes
    +NA
    RNA
    4rotein
    %or&
    Central +ogma" o) mol iol
    +NA
    RNA
    4rotein
    %or&
    Transcription:
    A !omple9 o) proteins
    re!ognise a promoter
    se6$en!e" at the start o)
    a gene
    This protein !omple9
    re!r$its RNA polymerase
    RNA polymerase $ses
    +NA as a template to
    ma&e a !omplementary
    strand o) RNA
    RNA )loats away )or
    pro!essing000
    +NA is le)t $n!hanged
    Central +ogma" o) mol iol
    mRNA is a template )or protein
    man$)a!t$re
    mRNA )eeds thro$gh a ribosome*
    whi!h re!r$its protein s$$nits
    #$$nits get assemled into peptide
    !hain* $ntil end o) the mRNA is
    rea!hed
    +NA
    RNA
    4rotein
    %or&
    Central +ogma" o) mol iol
    4rotein )olds into !omple9 shape* ready )or
    )$n!tion
    +NA
    RNA
    4rotein
    %or&
    H$ge range o) potential )$n!tions=
    Central +ogma" 5 re!ap
    +NA , !entral in)ormation store
    ...transcribed to...
    RNA , wor&ing !opy* a!ts as a template
    ...translated to...
    4rotein , the !ompleted ma!hine
    +NA
    RNA
    4rotein
    %or&
    Central +ogma" , >r000 sort
    o)000

    All the aove is !orre!t000 $t very in!omplete=


    +NA se6$en!es !an e str$!t$ral* reg$latory or
    !oding 2or any !omination o) these=3
    RNA se6$en!es !an e str$!t$ral* reg$latory*
    enzymati!ally a!tive* !oding 2or !ominations o)
    these=3
    (eta5in)ormation/
    #pli!e variants
    ?o!alisation signals
    (odi)i!ation signals
    000et!* et!000

    However* this is good eno$gh )or most !loning


    wor&=
    +NA
    RNA
    4rotein
    %or&
    Introd$!tion to !loning
    Cloning is 2$s$ally3 NOT ao$t
    ma&ing !opies o) whole
    organisms=
    I &now* I was disappointed too000
    Introd$!tion to !loning

    The aim o) !loning is to end $p with a


    plasmid that we !an p$t into o$r target
    organism* where it will e))i!iently e9press
    o$r desired gene0

    @ses in!l$de/

    protein prod$!tion 2e0g0 Ins$lin man$)a!t$re3

    improved s$rvival 2e0g0 Antiioti! resistan!e3

    Red$!ed s$rvival 2new v$lnerailities to dr$gs3

    modi)ied ehavio$r 2new rea!tions to stim$li3

    Creating )$sion proteins

    000et!
    scAAV-LP1-OligoN
    3687 bps
    500
    1000
    1500
    2000
    2500
    3000
    3500 NotI
    ITR
    HCR
    AAT
    'PolyA
    ITR
    amp
    Introd$!tion to !loning
    Restriction Enzymes
    re!ognise spe!i)i! short
    +NA se6$en!es* !$t the
    +NA at that point
    Sticky ends 1 overhangs
    allow +NA )ragments to
    e ligated together
    hFIX-514
    7966 bps
    1000
    2000
    3000
    4000
    5000
    6000
    7000
    NotI
    NotI
    hF
    Plasmids are !ir!les o) +NA
    that !an e p$t into many
    !ell types 2a!teria* yeast*
    animal* plant* et!0003
    %e !an $se them to !arry o$r
    gene o) interest into a
    target organism 2typi!ally
    a!teria* yeast or a !ell
    line3
    Introd$!tion to !loning
    >le!trophoresis allows $s to
    separate* vis$alise and
    e9tra!t desired )ragments*
    ready to p$t the right ones
    together000
    Introd$!tion to !loning
    Trans)e!t 1 Trans)orm 1 Transd$!e plasmid into yo$r !ells
    Then need to sele!t )or !ells that have s$!!ess)$lly ta&en $p the
    gene
    (ost !ommon approa!h is antiioti! sele!tion
    >nd res$lt/ a p$re !$lt$re o) easties e9pressing the novel
    !omination o) gene2s3 yo$Ave !hosen* in the !onditions yo$Ave
    designed
    Core te!hni6$es
    Polymerase Chain Reaction
    A method to rapidly !reate many !opies o) a +NA se6$en!eB
    essential )or modern mole!$lar iology
    Two primer" se6$en!es are
    mi9ed with the template +NA* a
    +NA polymerase* and dNT4s0
    The temperat$re is !y!led to
    progress the rea!tionB ea!h
    !omplete !y!le do$les the
    n$mer o) !opies present
    Res$lting +NA !an e $sed )or
    dete!tion* 6$anti)i!ation* !loning*
    m$tagenesis* et!000
    Core te!hni6$es
    Variants on the PCR:
    RT54CR to dete!t RNA strands
    64CR 2or RT564CR3 to !o$nt n$mer o) strands
    ($tageni! 4CR , to !reate da$ghter" strands in!l$ding a new
    m$tation
    Core te!hni6$es
    Western blotting and E!S"
    Antiodies ind to and
    re!ognise spe!i)i! proteins
    Can e $sed to immoilise yo$r
    proteins or -$st to dete!t them
    (a&e yo$r own 2hard* time
    !ons$ming* $nethi!al and very
    illegal=3 or !ommer!ially
    availale
    Core te!hni6$es
    Western blotting and E!S"
    These te!hni6$es $sed to
    !on)irm or meas$re
    e9pression o) yo$r protein
    >le!trophoresis is $sed to
    separate proteins y size
    Then protein smear" is
    proed with antiody to
    dete!t yo$r spe!i)i! targetB
    sho$ld all e at the same
    height on the gel
    >?I#A is similar* $t no
    ele!trophoresis000
    Core te!hni6$es
    #btaining and checking $%"
    (any tho$sands o) annotated gene se6$en!es availale )or
    )ree* online 2start at http/11www0n!i0nlm0nih0gov13
    +NA made to order is !onstantly getting !heaper000 7C<p 1
    ase
    ?imitations on length o) !ommer!ial +NA synthesis* $t
    wor&aro$nds e9ist
    >))i!ient se6$en!ing is hard000 $t !heap to o$tso$r!e0 (any
    dedi!ated servi!es to do it y post 1 drop in0
    Key tools D reso$r!es )or a
    io la

    >nvironment/ !leanliness* !ontainment*


    disposal

    Reagent handling/ need to meas$re tiny


    vol$mes 1 masses

    Centri)$ge

    Thermal !y!ler

    >le!trophoresis tan& 2D power s$pply3

    In!$ator D water ath


    Key tools D reso$r!es )or a
    io la
    Pipettes
    Handling a wide range o)
    vol$mes* some in sterile
    !onditions
    Key tools D reso$r!es )or a
    io la
    Centri&'ge
    Need to spin samples )or separating
    layers o) !hemi!als* pelleting +NA
    )or p$ri)i!ation* $sing vario$s la
    &its000 An essential tool0
    #ensile minim$m is to sa)ely spin a :
    gram sample at CC*;;;g
    - A!hievale with a dremel)$ge"* i)
    yo$ get someone else to hold it B3
    ?arger masses are desirale 2e0g0
    <;ml or <;;ml t$es $p to CC*;;;g3
    $t very !hallenging to do sa)ely0
    >normo$s energies involved=
    Key tools D reso$r!es )or a
    io la
    Electrophoresis tank
    #eparating +NA* RNA or protein
    a!!ording to size1!harge ratio0
    B$))ers are $sed to give
    everything in the sample the
    same !harge* then samples are
    dropped into a matri90
    Eoltage is applied* )or!ing the
    2!harged3 )ragments to migrate0
    #peed 2and there)ore distan!e3 o)
    migration is determined y
    )ragment size0
    Key tools D reso$r!es )or a
    io la
    !nc'bator ( Water
    bath
    Eario$s organisms strongly pre)er
    parti!$lar temperat$res and sho$ld e
    grown in a )airly narrow temperat$re
    range0
    As a !onse6$en!e* many enzymes 2esp
    restri!tion enzymes and polymerases3
    are very ine))i!ient o$tside narrow
    temp ranges0
    There)ore* need somewhere warm5$t5
    not5hot to grow $gs and r$n
    rea!tions=
    2(ost $gs and enzymes in reg$lar $se
    are est at 8F0<G3
    Key tools D reso$r!es )or a
    io la
    Thermal cycler
    >ssential )or 4CR
    Basi!ally -$st a programmale
    hotplateB a typi!al programme is/
    #TART/
    H<G )or C; min$tes
    +o I; times J
    H<G )or C min
    <I0<G )or C min
    K;G )or : min
    L
    K;G )or C; min
    >N+
    M;0<G is a sensile target000
    openp!r0org
    Key tools D reso$r!es )or a
    io la
    Spectrophotometer
    (eas$ring !olo$r !hange to tra!&
    rea!tions* meas$re protein
    prod$!tion* et!0
    Key tools D reso$r!es )or a
    io la
    Reagents
    A lot o) mole!$lar iology !an e done with &its* whi!h streamline pro!esses
    and avoid nasty !hemi!als000 >9pensive
    A lot o) wor& going into avoiding nastier and more e9pensive !hemi!als within
    the !omm$nity
    Care)$l planning 2e0g0 Biori!&s3 !an minimise the ne!essary reagent lirary
    Restri!tion enzymes are a ig !hallenge
    The !omm$nity needs to settle on model organisms to wor& with000 Eersatility
    and sa)ety are !on!erns=
    Key tools D reso$r!es )or a
    io la
    Sa&ety E)'ipment
    (ost o) modern mole!$lar iology is pretty sa)e i) yo$Are !are)$l0
    (ain hazards/
    4ower so$r!es 2$p to :;;E* I;;m%3
    Centri)$g$es , t$es or entire rotors )lying o)) i) imalan!ed=
    A )ew nasty !hemi!als 2a!ids* al&ilis* m$tagens3 , generally OK
    with gloves* la !oat* goggles and wor&ing in a well5ventilated
    area
    B$rns wor&ing with $nsen )lames
    000and the organisms yo$Are wor&ing with=

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