LAB 9) Bacterial Transfromation

Escherichia Coli

• E. Coli

• Kingdom Eubacteria

• Microscopic, prokaryotic singles celled organism

• Only visible through compound microspcoes

  • 1-2 microns long

• Duplicate in 20 mins

Bacterial Transformation

• Used for research and medicine

  • DNA cloning for protein insulin

pGLO

• A recominant plasmid

  • Bacterial DNA + Jellyfish DNA

• For genetically modified E.coli

• Expression vector

• Recombinant DNA molecules

• Contain DNA from more than one organism

Plasmid

• A small circular piece of DNA

  • Replicated by cells DNA and RNA polymerase

• Floating in cytoplasm

Expression Vector

• Plasmid used to express a particular gene

pGLO genes

• GFP gene

  • Glow green under UV light

• Beta-lactamase

  • Protects agains antibiotic ampicillin

• Protein araC

  • metabolism of sugar arabinose

Biotechnology

• Uses living organisms to make biological products

• Manipulate genetic material and fusion of cells not possible in nature

  • Gene therapy, plant cell culture, and recombinant genetic engineering

GMO’s

• An organisms with altered genetic material

• Create functional products that non-GMo would not normally produce

• INSULIN

Insulin

• The 1st licensed drug produced by GMO

Genetically Modified Crops

• Genetically modified bacteria transferred into plant cells

Genetically Modified Bacteria

• Genetic info in nuleoid (NON MEMEBRANE BOUND)

• Some have Plasmid

Steps of Bacterial Transformation

1. Extract plasmid DNA

2. Insert gene of interest into plasmid

EXTREMELY INEFFECTIVE

Gene Expression

• Gene is expressed when it has been converted from DNA to protein

• Gene expression controlled by promoter

Promoters

• Bind RNA polymerase to the DNA for transcription into RNA

• Binding can be regulated by regulatory proteins

Regulatory proteins

• Saves energy when expression of gene isn’t necessary

• Bind near the promoter of the gene to physically prevent RNA polymerase from binding to the promoter to transcribe the gene into RNA

Unique sequences of pGLO

1. DNA sequence for plasmid to replicate in E.Coli

2. DNA sequence that encode for the 3 genes of interest

3. DNA sequence for promoters that control expression of each gene of interest

bla

• Genes that encodes for protein beta-lactamase

  • Breaks down the antibiotic ampicillin

• Controlled by a NON REGLATED E.Coli promoter

• Selective gene

Selective Gene

• Bacteria with bla gene will grow in the presence of antibiotics

• So we know if they have transcribed successfully if they even survive.

• Good since transformation is EXTREMLY ineffective

araC

• Gene that encodes for preotin AraC

  • Metabolism of arabinose

  • AraC (protein) regulates the promoter P_BAD which controls expression of the genes that metabolize arabinose

• Controlled by a NON REGULATED E.Coli promoter

gfp

• Gene that encodes for green fluroecent proetin

• Controlled by REGULATED promoter P_BAD

• Does not need any co-factor

  • only UV light (40 - 400 nm)

  • b/w x-rays and visible light

• REALLY small 238 amino acids

  • Can be used as a tag

P_BAD

• A regulated promoter

• Control expression of araB, araA, and araD

When no arabonose

• The regulatory proetin AraC binds to DNA in P_BAD promoters and inhibit RNA polymerase from binding to the promoter

• AraC inhibits the expression of the genes that P_BAD controls

When arabinose is present

• Arabonose binds to Arac and changes the proetin

• AraC-arabinose complex allows RNA polymerse to bind to P_BAD

PGLO metabolism gene

• Replaces with gfp

• The presence of arabinos = expression of gfp gene

Lateral gene flow

• Natural transformation

Calcium Chloride induced bacterial transformation

• EXTREMELY INEFFECTIVE

1. Exposes bacteria to Ca2+ ions

   1. Neutralize the negatively charge phosphate groups AND plasmid

   2. become COMPONENT CELLS

2. Heating the bacteria (42 C) and cooled at (0 C) recover at (37 C)

   1. Opens the cells pores (adhesion zones)

   2. Plasimid on surface of the cell are moves into the cells cytosol during heating

Luria Broth

• On the plates

• SOme have ampicillin and or arabinose

Dilution Factors

• 10,000 and 1,000,000

Origin of replication

• !st site of numbering a plasmid

• ALlos the plasmid to be replicated in E.coli by DNA polymerase

Steps to explain how the gene gfp gets made into protein product GFP

1. Arabinose binds to the AraC protein

2. RNA polymerase is recruited to bind to the PBAD promoter

3. RNA polymerase transcrived gfp DNA into mRNA mRNA is translated by cytosolic rivosomes into the GFP protein

4. GFP is exposed to UV light and glows green.