biotech and lab exam - bio 211

plasmid features

• ori - where replication starts, spot where replication factors bind to

• selection marker - helps to mark bacteria with gene inserts

• ARG - antibiotic resistant marker, makes sure only bacteria with gene inserts survive

• MCS - site where gene insert is inserted

cloning plasmid

stores genes and replicates, but doesn’t express them

expression plasmid

contains promoter, plasmid cloning site (shine-Dalgarno site) for transcription and translation of the gene insert

restriction enzymes

recognize and cut the 5’ end and the 3’ end of plasmids and the gene of interest, creating sticky ends

• allows for directionality of transcription and translation, helps them to fit together

clone insertion steps

1. identify the palindrome recognition site in the plasmid and the gene (same sequence in both directions)

2. use restriction nuclease to cut ends on both

3. insert gene into plasmid via sticky ends

4. insert into bacteria

transformation

the process of inserting a plasmid into a host cell/organism

• allows the host to clone/express the gene as a protein, allowing for phenotypic observation

components required for pcr

• TAQ polymerase

• dNTPs

• forward and reverse primer

• DNA template (plasmid, genomic DNA, cDNA)

• buffers and salts or divalent ions

• thermal cylinder

primer requirements

• 18-30 nucleotides long

• 30-60% GCs

• cannot be self-complimentary (forming secondary structure) or complimentary to the primer in the opposite direction (forming a primer dimer)

forward v reverse primers

• forward primer is complimentary to one strand and is UPSTREAM of target DNA

• reverse primer is complimentary to the other strand and is DOWNSTREAM of target DNA

PCR steps

1. dsDNA is heated to 95 degrees, separating strands

2. temp is reduced to 55 degrees, allowing primers to anneal to opposite strands in oppostite directions

3. temp is increased to 72 degrees and dNTPs are added

4. process repeats 30-35 times, producing millions of clones of the desired fragment

genomic DNA library formation

1. restriction enzyme cuts random portions of the genome, producing equally sized fragments of DNA (includes introns, non-coding portions)

2. fragments are inserted into plasmids

3. plasmids are inserted into bacteria

used in gene cloning

cDNA library formation

1. isolate mRNAs from other types of RNA with oligo-dT columns via binding to poly A tails

2. use reverse transcriptase to synthesize cDNA from mRNA

3. use DNA pol to create a complimentary strand of DNA (to make it double stranded)

4. insert cDNA into a vector or plasmid

5. transform/insert plasmids into bacteria

used in gene expression

cDNA

• doesn’t contain introns

• reveals abundance of mRNA (the amount/ratio of cDNA produced is based on the mRNA in the cell)

• reveals splice isoforms (pattern) used, because only exons are shown

genomic DNA

• contains introns

• doesn’t reveal mRNA abundance or splicing patterns

Sanger v. PCR

• — uses a high fidelity polymerase with the ability to proofread, — uses taq, which is error prone

• — uses ddNTPS and dNTPs, pcr only uses dNTPs

• — uses one primer, pcr uses two that work in opposite directions

• — produces lots of different sized fragments, — produces millions of copies of one size of fragment (clones)

components required for sanger sequencing

• one primer (sr1 or sr2)

• heat stable DNA polymerase

• lots of dNTPs

• a bit of ddNTPs (fluorescently marked)

• DNA template

• buffers and salts

how does sanger sequencing reveal nucleotide order?

• dNTPs and ddNTPS are added to the DNA template randomly

• each fragment is a different size and is used to determine the sequence

• fragments are separated by capillary gel

• laser and detector form a chromatograph based on fluorescently marked ddNTPS

CRIPR full name

clusters of regularly interspaced short palindromic repeats

cas9

a nuclease that interacts with guide RNA and targets viral DNA

• cleaves/removes viral DNA to protect bacteria

CRISPR-CAS9

• viral DNA is inserted into the CRISPR locus

• RNA polymerase transcribes CRISPR locus, producing crDNA

• crDNA forms a complex with Cas 9, helping it to target and cleave matches

how can ds breaks from CRIPR-CAS9 be fixed

non-homologous end joining: error prone, glues together ends after viral DNA is cleaved

homologous repair: error-free, requires a sister chromatid

how can CRIPR be used in gene editing

• removal of mutated DNA at specific sites, minimizing side-effects and maximizing positive results

• mutated DNA could be replaced with desired DNA

features of c. elegans

• full life cycle is complete in 3 days

• transparent, allowing us to see in-vitro via fluorescent markers

• can reproduce independently or with other males

• can be fed simple diet (bacteria)

• cheap, easily accessible, small

c elegans life cycle

embryo

L1

L2

L3

L4

adults

• sexes diverge at L4 stage

• after L1, worms may enter dauder stage due to limited resources or poor environmental conditions

• L4’s used to test phenotypes and in RNAi

how can c elegans propagate

• hermaphrodites can self-fertilize (produce sperms and eggs)

• males can fertilize hermaphrodites to introduce mutations

what can be seen on worm plates

all sizes of worms:

• adults are large and thick

• L4s have characteristic white crescent marking

• L3, 2, and 1 are all small

• small, circular embryos may be seen

• dauder are thin and long

info on wormbase

• functions of genes and proteins and their phenotypes

• location of genes in an organisms genome

• length of coding sequences for genes

• where a protein is expressed and how it interacts with other genes

• homologous genes in different organisms

RNAi steps

short for RNA interference

1. dsRNA is formed via transcription and becomes fragmented by Dicer

2. siRNA and microRNA are formed

3. siRNA and microRNA are unwound and bind to complimentary mRNA

4. dsRNA is cut out from other sections and degraded, preventing it from being expressed

how is RNAi delivered to worms

• via injection (most precise)

• via feeding (we used bacteria with dsRNA incorporated, easiest)

• via soaking in solution containing dsRNA (difficult to form dsRNA in-vitro)

RNAi trigger

• for our project, we used IPTG, which activated our plasmid

• otherwise, viruses or any other addition of dsRNA can — this pathway

L440 plasmid

• vector made up of the ori, MCS, Amp, and T7 promoter

ori

dictates where and when a plasmid replicates

• replication factors bind here to begin transcription and ensure that the gene insert is replicated

MCS (multiple cloning site)

where the chosen gene is incorporated into the plasmid

AMP

ensures that all bacteria hold the chosen gene and can help to prevent contamination

t7 promoter

inverted and transcribe DNA strands at the same time, forming dsRNA

Ahringer library

made up of 16 000 RNAi clones that target c. elegans

• each clone is made up of the L4440 vector and a gene insert

how to create a single colony from a clone

1. use a pipette tip to remove bacteria from the container the clone is in, swirling around to ensure the tip is coated

2. gently rub over ¼ of the LB agar plate

3. Repeat three times to fully streak the plate

can be stored at 37 degrees for growth or 4 degrees for safekeeping

• one — is necessary to ensure that all progenies are identical and contain the same mutations (if any)

positive control

a — with a known phenotype that’s used to determine if an experiment is running as expected

• we used GMC101

negative control

a — that doesn’t produce a response and is used to determine if an experiment is reliable

• we used N2 and smd-1

what makes GMC101 unique

• its a positive control

• exhibits full 1-42 amyloid beta found in humans

• results in C. elegan paralysis when incubated at 25 degrees and higher

how we did our first paralysis test

• GMC101 and N2 strains were placed on separate plates and incubated at 20 and 26 degrees

• tested for paralysis at 48h and 72h marks

• GMC101 should exhibit full paralysis, N2 shouldn’t exhibit any

why GMC101

it produces the full amyloid beta strand produced in humans, making it a good model for Alzheimer’s and how these clusters affect the brain

• if chosen gene has an impact on how GMC101 aggregates (decreases or increases paralysis) it can help us determine what to study further

buffers used in plasmid purification

P1, P2, N3, PE, EB

P1

made up of RNase, glucose, Tris, EDTA

• breaks down cellular RNA, prevents DNA from degrading

P2

made up of sodium hydroxide, SDS

• breaks down cell walls, denatures proteins, forms holes in cell plasma membranes

N3

made up of potassium acetate

• increases cell pH

PE

contains ethanol

• used to remove salts and contaminants from solution

EB

made up of Tris-cl

• used to isolate DNA from all other substances

how are proteins and membranes separated

initially separated by P2 buffer

• process continues as buffered are added to column and its spun, leaving DNA in the tube (as supernatant) or in the column as the solution is spun

agarose gel

made up of agarose powder and TAE buffer

• found in seaweed, forms mesh-like substance when heated and cooled

• TAE is used to dissolve agarose powder and helps to maintain a neutral pH

gel electrophoresis explained

• samples of gene sequences are inserted beside DNA ladder at the negative end of the electrophoresis box

• current causes DNA to move towards the positive end (because DNA is negatively charged)

• shorter strands move faster than longer strands

• quality is determined by defined/distinct bands

Gel green

incorporated into agarose gel or gel can be dipped in it before UV expsure

• binds to DNA and produces florescence that makes bands visible

loading dye

incorporated into DNA solutions before loading into wells

• allows us to see movement of substances through the gel

• keeps DNA grounded in wells