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

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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rasromeo
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© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
448 views

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

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.

Uploaded by

rasromeo
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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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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