vectors and libraries

Why couldn’t scientists purify genes like they did proteins?

They purified proteins by charge and size, but dna is made up of a c t and g which are very similar in terms of size and charge. Genes are also in chromosomes, which break super randomly and easily often in the middle of genes.

How did they purify individual genes?

By inserting dna fragments of interest into vectors, then cloning the vectors by replicating the bacteria

What is the vector type used in Waksman

Plasmid

What is the name of the plasmid used in Waksman

pTriplEx2

How did they insert the gene of interest into ptripleex2 (digest then ligate)

They used restriction enzymes (sfil) to cleave both the insert and vectors at a (sfila and sfilb sites)

Then added dna Ligase to Ligate the insert and vector

How did they transform and amolify

When a bacteria replicates, its plasmid is replicated regardless if it is its own dna.

Plasmid structure of manmade ones

Origin of replication (pUC ori) the replication proteins in bacteria bind to ori, and initiate replication of plasmid.

Selection marker (ampr and lacz genes) that encode proteins that allow us to see which bacteria have the plasmid

Cloning site (MCS) which is a stretch of dna between the lacz gene, acting as a site to put our insert and disrupt the lacz gene.

Why can’t plasmids be directly put into the bacteria?

Plasmid dna has a negative charge (phosphate in backbone) and repels the negative charge of the bacteria membrane (phosphate group in phospholipids)

How do we make bacteria more permeable to plasmids

Treat it with cacl2, and the ca2+ will bind to the membrane and make it more positively charged, allowing it to attract to the plasmid

How do u identify the cells with the plasmid?

The ampr gene makes it immune to ampicillin, so the ones w plasmid will live and grow.

How do u identify cells w the insert

If an insert is present in the mcs, the lacz gene will be distrupted. The lacz gene usually codes for beta galactosidase enzyme which cleaves the substrate xgal (present on agar), leaving the substrate blue. If disrupted, the lacz gene will code for a truncated beta galactosidase enzyme, and therefore the xgal will stay intact and be white.

Landolita punctata

Duckweed, the goal of Waksman is to identify expressed genes in duckweed.

DNA library

A random collection of dna fragments from an organism with ideally one copy of every dna sequence

The dna fragments in libraries are cloned into vectors

They are easily maintained in a lab

Can be manipulated to isolate dna fragments of interest (just isolate plasmid with the fragment)

Genomic library

Contains all randomly generated dna fragments from genome: all genetic material, coding and noncoding, promoters, introns, intergenic

cDNA library

Contains only expressed dna that codes for proteins

Cdna is complimentary dna (copy of mRNA in the form of dna)

U can’t use mRNA as libraries because they are single stranded, unstable, and cannot clone and are difficult to sequence.

Partial digest

Partial restriction enzyme digest prevents the enzyme from cutting the target gene at every site, then u isolate multiple fragments that may have overlap so u can get the full gene.

1. Isolate the rna

Freeze cells in liquid nitrogen, grind using a mortar to release cell contents, centrifuge to pellet debris, use ethanol and licl To precipitate rnas

2. Purify the mRNA

Pass the rna over an oligo dT column, the poly a tails in mRNA will hybridize the mRNA to the column, then wash away the unwanted rna, then elite the mRNA to get pure mRNA

3. Reverse transcribe mRNA

3. Use reverse transcriptase, uses the T tail as a primer, synthesizes complimentary dna strand, then adds c to very end through terminal transferase activity, results in dna rna hybrid

4. Remove rna from rna dna hybrids

4. Add a strong base like Naoh to hydroloze the rna strand (it’s more susceptible than dna due to its OH at 2 prime carbon)

5. Perform second strand dna synthesis

5. Anneal oligonucleotide to the string of c at end of dna, dna polymerase uses that to synthesize dna

6. Ligate double strand cdna into pTriplEx2 vector

6. Use sfil to cut vector and cdna, cdna allows the plasmid to circularize

7. Transform bacteria

Add cacl2 to bacteria, add to plasmids, then incubate to let them in. Place bacteria in lb agar to replicate ( then use selectable markers to choose colonies)