Ara Operon Notes_18.docx

pGLO plasmid and L-arabinose operon The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jellyfish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism express its fluorescence under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.

pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows: i)

bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family) ii) araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose) iii) GFP, which codes for the green fluorescent protein, that gives a green glow if cells produce this type of protein

Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The pGLO plasmid was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba.

Other features on pGLO, like most other plasmids, includes: a selectable marker, ori (origin of replication), and an MCS (multiple cloning site) located at the end of the GFP gene. The plasmid is 5371 base pairs long.

Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.

For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"form, or structurally analogous to D-glyceraldehyde. However, L-arabinose is in fact more common than D-arabinose in nature and is found in nature as a component of biopolymers such as hemicellulose and pectin.

The L-arabinose operon, also known as the araBAD operon, has been the subject of much biomolecular research. The operon directs the catabolism of arabinose in E. coli, and it is dynamically activated in the presence of arabinose and the absence of glucose. In synthetic biology, arabinose is often used as a one-way or reversible switch for protein expression under the PBAD promoter in E. coli. This on-switch can be negated by the presence of glucose or reversed off by the addition of glucose in the culture medium which is a form of catabolite repression.

The L-arabinose operon, also called the ara or araBAD operon, is an operon that encodes enzymes needed for the catabolism of arabinose in Escherichia coli. It has both positive and negative regulation and is activated allosterically. It has been a focus for research in molecular biology since 1970, and has been investigated extensively at its genetic, biochemical, physiological, and biophysical levels. In E. coli, arabinose is converted to xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The structural genes, which encode enzymes for arabinose catabolism, are araB, araA, and araD (collectively known as araBAD). The regulator gene is araC. The genes araBAD and araC are transcribed in opposite directions.

i)

araA encodes L-arabinose isomerase, which catalyses isomerization between L-arabinose and L-ribulose. ii) araB encodes ribulokinase, which catalyses phosphorylation of (L/D)-ribulose to form (L/D)ribulose 5-phosphate. iii) araD encodes L-ribulose-5-phosphate 4-epimerase, which catalyses epimerization between L-ribulose 5-phosphate and D-xylulose 5-phosphate. D-xylulose 5-phosphate and D-ribulose-5-phosphate are metabolites in the pentose phosphate pathway. The operators are araI and araO2.

AraI lies between the structural genes and the operator. The araI1 and araI2 are DNA-binding sites that, when occupied by AraC, induce expression.

Repression – The ara operon is regulated by the AraC protein. If arabinose is absent, the dimer AraC protein represses the structural gene by binding to araI1 and araO2 and the DNA forms a loop. The loop prevents RNA polymerase from binding to the promoter of the ara operon, thereby blocking transcription.

Activation – When arabinose is present, arabinose binds AraC and prevents AraC from interacting. This breaks the DNA loop. The two AraC-arabinose complexes bind to the araI1 and araI2 sites which promotes transcription. When arabinose is present, AraC acts as an activator.

Metabolism of arabinose in E. coli Substrate

Protein(s)

Function

Reversible Product

L-arabinose

AraA

isomerase

yes

L-ribulose

L-ribulose

AraB

ribulokinase

no

L-ribulose-phosphate

L-ribulose-phosphate

AraD

epimerase

yes

D-xylulose-phosphate

If arabinose is present, it builds a complex: AraC + arabinose This complex is needed for RNA polymerase to bind to the promoter and transcribe the ara operon. Also for activation the binding of another structure to araI is needed: CRP (formerly known as CAP) + cyclic AMP So the activation depends on the presence of arabinose and cAMP.