Transformation experiment on E. coli with pGLO plasmid to elucidate arabinose metabolism.

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Transformation Experiment on E. coli with pGLO | M4S Digital Textbook
M4S Microbiology for Students
Molecular Biology • Digital Learning Chapter

Transformation Experiment on E. coli with pGLO Plasmid

A structured M4S learning chapter covering plasmids, transformation, the arabinose operon, GFP, pGLO characteristics and interpretation of the supplied transformation results.

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A plasmid is a piece of extrachromosomal DNA present in bacteria that is capable of self-replication. The supplied source describes plasmids as genetic tools that can be introduced into bacteria through transformation and used to express genes and produce proteins.

Key point: The source identifies fertility F-plasmids, resistance plasmids, virulence plasmids, degradative plasmids, and col plasmids as commonly known plasmid types.

Genetic transformation refers to the introduction and expression of foreign DNA in a host organism. Escherichia coli is widely used for genetic transformation. DNA uptake and translocation can result in transformation.

The supplied source describes making cells competent as a common approach. “Competent” refers to the ability to absorb DNA from the surrounding environment, and the source states that treatment with calcium chloride can make E. coli cells competent.

Bacterial transformation illustration
Bacterial transformation illustration from the supplied source.

An operon is a collection of genes transcribed into RNA by a single promoter. The arabinose operon is an example involved in metabolism of the sugar arabinose.

The supplied source identifies araB, araA, and araD as clustered genes involved in arabinose degradation, with transcriptional initiation from the pBAD promoter. It also describes the involvement of RNA polymerase, AraC, and arabinose in transcription.

Without arabinose: the source states that AraC binds near the promoter and prevents transcription of the digestive enzymes.
With arabinose: the source states that arabinose binds AraC, causing a conformational change and allowing expression of araB, araA, and araD.

Green fluorescent protein (GFP) is a protein of approximately 28 kDa that emits green fluorescence when exposed to ultraviolet light. The source states that GFP was isolated from the marine jellyfish Aequorea victoria.

The gfp gene is widely used as a reporter in cellular and molecular biology to visualize gene expression, including expression following genetic transformation. GFP can also be used to mark organelles, cells, and tissues.

Reporter principle: the source describes green fluorescence as an indicator that the cell expresses the introduced GFP-linked construct.

The pGLO plasmid contains DNA sequences that enable replication and expression of GFP in bacteria after transformation. The supplied source identifies several important elements:

ComponentRole described in the source
GFPReporter gene responsible for green fluorescence.
oriReplication origin.
blaSelectable marker identified in the pGLO plasmid.
pBAD promoterPromoter associated with arabinose-regulated expression.
araCGene encoding the arabinose C regulatory protein.
Cloning sitesSites included among the plasmid's DNA elements.

The source also notes that reporter genes such as GFP have advantages related to stability but may have artifacts.

pGLO transformation experiment results
Photograph 1: Transformation experiment. Left: green colonies after UV illumination on LB/AMP/Arabinose with plasmid; right: white colonies after UV illumination on LB/AMP with plasmid. Picture credit: Mr. Roshan Timsina (MSc, Public Health Microbiology).

Green colonies

In the supplied photograph description, green fluorescence is associated with the LB/AMP/Arabinose condition containing plasmid.

White colonies

In the supplied photograph description, white colonies are associated with LB/AMP containing plasmid without the stated arabinose condition.

Interpretation: the supplied material links arabinose-dependent regulation of GFP expression with the observed difference in colony fluorescence.
  1. Alberts B, et al. Molecular Biology of the Cell. 6th ed.
  2. Frontiers in Microbiology DOI: 10.3389/fmicb.2015.00242
  3. IntechOpen — Plasmids as genetic tools and their applications in ecology and evolution
  4. IntechOpen — supplied duplicate reference
  5. Journal of Bacteriology PDF
  6. DOI: 10.3791/253
  7. DOI: 10.1016/B978-012253840-7/50005-5
  8. DOI: 10.1038/newbio233166a0
  9. DOI: 10.1111/j.1574-6976.2010.00226.x
  10. DOI: 10.1016/B978-012253840-7/50005-5
  11. DOI: 10.1038/newbio233166a0
  12. DOI: 10.3791/253
  13. DOI: 10.1111/j.1574-6976.2010.00226.x
Transformation Experiment on E. coli with pGLO Plasmid • M4S Digital Textbook • Prepared by M4S

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