3 molecular cloning/transformation/transfection

molecular cloning

produce large amounts of recombinant DNA

recombinant DNA

combining DNA from different sources

insert

gene/DNA want to clone

obtained by PCR amp

vector

DNA molecule used to carry the insert, usually a plasmid

applications of cloning

1. protein production (ex. growth hormones, used in research)

2. obtain large quantities of DNA (sequencing, gene structure studies, gene regulation)

3. modify phenotype/genotype (ex. insert gene → cell expresses new protein)

molecular cloning steps

1. Plasmid purchase/preparation

2. Generate monoclonal bacteria: streak plate, isolate single colony, grow liquid culture

3. DNA prep: isolate plasmid DNA

4. Cloning: digest, ligation

5. Transformation

6. Sequencing

7. Glycerol stock: long term bacterial storage

DNA prep/plasmid isolation

miniprep: small DNA yield

midiprep

maxiprep

transformation

plasmid inserted into bacteria

heat shock, electroporation

DNA ligase

enzyme that joins DNA fragments by forming phosphodiester bonds

result: recombinant plasmid

screening colonies

identify bacteria with correct insert

methods: colony PCR, restriction digest, sequencing

restriction enzyme/digest

enzyme that cuts DNA at specific recognition sequences

digest: create compatible ends for ligation

choosing restriction enzymes

check plasmid MCS (make sure enzyme cuts in MCS)

ensure enzyme does not cut gene (or else fragmented)

orientation (2)

plasmids

small, circular, double-stranded DNA

high copy number

many plasmids per bacterial cell → lots of DNA

sticky ends

DNA overhangs produced by staggered restriction enzyme cuts that facilitation ligation

blunt ends

DNA ends with no overhangs produced by straight restriction cuts

origin of replication (ori)

DNA sequence allowing plasmid replication in host cell

selection marker

gene allowing identification of transformed cells, usually antibiotic resistance

promoter

DNA sequence that initiates transcription of a gene

tag

protein sequence added to a protein to aid detection or purification

multiple cloning site (MCS)

region with many restriction sites, allows easy insertion of gene

PCR

method to amplify specific DNA sequences exponentially

denaturation

DNA strands separate 96C

annealing

primer bind complementary DNA sequence (50-65C)

extension

polymerase adds nucleotides to the primer’s 3′ end until it reaches the end of the template strand

72C

double digest

cutting DNA with two restriction enzymes simultaneously to control insert orientation

gel electrophoresis

separate DNA fragments by size

migrate to positive electrode

smaller fragments move faster (supercoiled, nicked)

gel extraction

cut DNA band from gel

purify DNA using kit

elute purified DNA

self-ligation

vector re-circularizes without insert during ligation

alkaline phosphatase

enzyme that removes 5’ phosphates from vector DNA to prevent self-ligation

ligation ratio

optimal molar ratio of insert to vector, typically 3:1

transformation efficiency

number of colonies formed per ug DNA

10^6-10^9 CFU/ug

factors affecting: plasmid size, DNA quality, competent cell quality

colony PCR

PCR screening method performed directly on bacterial colonies

blue-white screening

lacZ produces beta-galactosidase (inverts substrate into blue product), blue = plasmid without insert, white = with insert

positive selection cloning

method where only plasmids containing an insert allow bacterial survival

diff primers

insert-specific primers: bind within insert = band appears

backbone primers: bind vector sequences flanking (on both sides of) insert (checks insert by product size)

orientation primers: determine insert orientation; band only appears if insert is in the right direction (one primer in the vector, one in the insert)

plasmid conformations

uncut plasmids:

• relaxed (nicked) → slowest

• linear → medium

• supercoiled → fastest

diagnostic restriction digest

enzyme digestion used to verify insert presence and orientation

sanger sequencing

reads exact DNA sequence using chain-terminating nucleotides (ddNTPs)

PCR and cloning can introduce mutations

competent cells

bacteria capable of taking up foreign DNA

electroporation

DNA delivery method using electrical pulses to create membrane pores

pros: fast, broad cell compatibility

cons: high cell death

transfection

introduction of DNA into eukaryotic cells

transient expression

temporary gene expression where DNA does not integrate into the genome

stable expression

gene expression maintained after DNA integrates into the genome

biological transduction

delivery of genetic material using engineered viruses

piggyback

• 2 plasmids:

  • transposon (has your gene)

  • transposase (does the cutting/inserting)

• Mechanism:

  • transposase cuts out gene from plasmid (recognizing ITRs)

  • inserts it into genome (less specific, inserts at TTAA sites)

• Result:

  • stable integration (no virus needed)

synthetic biology

engineering biological systems to perform new functions

build from scratch, modify existing systems

biological circuit

network of interacting genetic components performing a programmed function

transcriptional unit

DNA region (promoter → RBS → gene → terminator) → transcribed into mRNA

BioBricks

standardized DNA parts that can be assembled modularly

2 combine → produce new BioBrick

uses isocaudamer restriction enzymes → create compatible sticky ends (different recognition sites)

ligation creates a scar sequence (cannot be cut again by those enzymes)

Idempotent assembly

assembly where the product remains compatible for further assembly (can keep adding parts)

Golden Gate assembly

cloning method using Type IIS restriction enzymes to assemble multiple DNA fragments in one reaction

pros: no scar sequences, one-pot assembly, multiple fragments joined simultaneously

→ programmable overhands