Biotechnology Exam Two

Slides from Chapter 3, 4, and 5.

Plasmid DNA: small circular pieces of DNA fiund primarily ion bacteria

small circular pieces of DNA found primarily in bacteria

Plasmid DNA facts:

• considered extrachromosomal DNA because they are in the cytoplasm in addition to bacteria chromosome

• are small, approx. 1 to 4 kb

• can replicate independently of chromosome

• used as vectors: pieces of DNA that can accept, carry, and replicate other pieces of DNA

Calcium Chloride Transformation of Bacterial Cells

• a process for inserting foreign DNA into bacteria

• very inefficient process

Calcium Chloride Transformation of Bacterial Cells steps:

1. Treat bacterial cells with calcium chloride, while chilled on ice for 30 minutes

2. Add plasmid DNA to cells chilled on ice

3. Heat the cell and DNA mixture (30 sec @42 degrees C)

4. Plasmid DNA enters bacterial cells and is replicated and express their genes

Electroporation

apply brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall that allow the DNA to enter the cell

Advantages of Electroporation:

• rapid

• requires fewer cells

• can be used to introduce DNA into other cell types

• more efficient process

Selection

is a process designed to facilitate the identification of recombinant bacteria from bacteria that contain plasmid without foreign DNA, while preventing the growth of non-transformed bacteria

Antibiotic selection:

• transformed cells are cultured on plates containing antibiotic to identify transformed bacteria from non-transformed bacteria

Ex.: Ampicillin if your plasmid has Amp. resistant gene

• does not select for plasmid containing foreign DNA (recombinant plasmid) vs. recircularized plasmid with no DNA insert

What is involved in the selection of recombinant bacteria after transformation:

• selection

• antibiotic selection

Blue-white selection with X-gal (artificial lactose) in media:

• DNA is cloned into plasmid restriction site within lacZ gene

• When lacZ gene is interrupted by an inserted gene, it cannot produce functional Beta galactosidase enzyme

• With X-gal added to media in Petri plate

-Non-functional lacZ = white colony = clone = genetically identical bacterial cells each containing copies of the recombinant plasmid

• With X-gal added to media in Petri plate

-Functional lacZ = blue colony

B. 1, 3, and 6

Introduction to human gene cloning:

• first recombinant human protein marketed was insulin in 1982, and next was growth hormone in 1985

• cloned human insulin cDNA sequence inserted into a plasmid and the bacteria cells were then used to synthesize the protein product of the cloned gene

• can generate lots of pure protein via this technique

What was the source of growth hormone prior to recombinant technology?

Cows (bovine) or pigs (porcine) provided insulin to treat human diabetes, then human growth hormone was extracted from the pituitary glands of human cadavers.

Practical Features of DNA Cloning Vector (Plasmid)

• Size

• Origin of replication (ori)

• Copy number

• Multiple cloning site (MS)

• Selectable marker genes

• RNA polymerase promoter sequences

• DNA sequence primers

Size

small enough to be separated from chromosomal DNA

Origin of replication

site for DNA replication that allow plasmids to replicate independently from host chromosome

Copy Number

number of plasmids in the cell, normally small (recombinant plasmids have high copy numbers)

Multiple cloning site (MCS)

recognition sites for several restriction enzymes in which DNA insert is cloned into

Selectable marker genes

enables selection of transformed colonies

RNA polymerase promoter sequences

used for transcription (for Protein Expression Vector (up arrow)) in vitro and in vivo

DNA sequence primers

seq flank ends of multiple cloning site

Assume you want to make lots of human insulin using a bacteria expression vector.

Why wouldn’t using human insulin genomic DNA for this cloning project be advantageous?

Because bacterial cells cannot remove introns, which are non-coding regions found within eukaryotic genes.

Creating DNA Libraries

• Collections of cloned DNA fragments from a particular organism contained in plasmid vectors within host bacteria (ex. E. coli), or can be flanked by ligated adapters/linkers (w/NGS) without use of host cells

• Screened to pick out different genes of interest

Two Types of Libraries:

• Genomic DNA libraries (Ex. DNA sequencing entire genome (NGS))

• Complementary DNA libraries (cDNA libraries) (Ex. Sequencing transcriptome)

What prevents restriction enzymes produced by a bacterium from digesting its own genome?

Methylated DNA

Genomic Libraries

• Chromosomal DNA from the tissue of interest is isolated & digested with a restriction enzyme (RE) which produces many fragments that are part of the entire genome

• DNA ligase is used to ligate genomic DNA fragments and vector (plasmids) digested with the same RE or adapters/linkers (w/NGS)

• Recombinant vectors (plasmids) are used to transform bacteria, and theoretically each bacteria will contain a single recombinant plasmid with a single fragment of genomic DNA from the organism of interest

Disadvantages of Genomic Libraries

• Introns are clones in addition to exons:

- Majority of genomic DNA is introns in eukaryotes so majority of the library will contain non-coding pieces of DNA

• Many organisms have very large genome, so searching for gene of interest is difficult, unless using NGS.

• Time consuming!

cDNA Libraries

• mRNA from tissue of interest is isolated

• Need to make double stranded DNA from mRNA; How?

• Adapters/linkers (w/NGS): double stranded cDNA sequences, which contain restriction enzyme recognition sites, are ligated to the ends of the cDNA

• Cut with restriction enzyme, cut vector with same RE, ligate fragments to make recombinant vectors

• Then transform bacteria with recombinant vectors, or perform NGS of cDNA with Adapters/linkers

cDNA Libraries

• Advantage over genomic libraries

- Collection of actively expressed genes in the cells or tissues from which the mRNA was isolated

-Introns are NOT cloned

-Can be created and screened using colony hybridization to isolate genes that are primarily expressed only under certain conditions in a tissue

• Disadvantage

-Can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available

Identify and Clone a Gene of Interest