Drybench Plasmid.
Ampicillin resistance: the bla gene and β-lactamase
The bla gene encodes β-lactamase, an enzyme that destroys ampicillin, so only bacteria that carry the plasmid grow on plates containing it.
The gene and the enzyme
pBR322's ampicillin resistance gene was among the first plasmid genes sequenced (Sutcliffe 1978). It encodes the TEM-1 β-lactamase, which opens the β-lactam ring of penicillins such as ampicillin and so inactivates them. The protein is made with a signal peptide that sends it out of the cytoplasm.
On a map, bla is usually drawn as an arrow on the bottom strand of pBR322 and pUC19. The library's copy comes from pBR322's own record and translates exactly to the sequence of its UniProt entry, so on pBR322 the coding part opens in the protein app; pUC19's copy differs from that entry at two residues, so on pUC19 it does not.
Selection
After transformation, cells are spread on medium with ampicillin: cells without the plasmid die, and colonies that grow carry it. Because the enzyme works outside the cell, it can lower the antibiotic around a colony, so plates are best read on time.
See it on a real plasmid
Open pBR322, pick AmpR (bla) and open its protein from the inspector.
β-lactamase is a finished protein doing its job, as the tour's last stop shows for haemoglobin.
References
- Sutcliffe JG. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A 1978;75:3737-3741. PubMed 358200
- Bolivar F, Rodriguez RL, Greene PJ, et al. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 1977;2:95-113. PubMed 344137
Statements summarised by the site's builder from the references, for scientific review.