IB Biology Year 1
KEY to the MolecuLab 115 Student Manual: Q’s for Comprehension
1. Most bioluminescent fish get their glow from bioluminescent bacteria living within light organs.
2. In the mutualism between bioluminescent bacteria and their fish or squid hosts,
 the bacteria are provided with a protected place to live and a supply of food,
 the fish use the light to ward off predators, attract prey, and/or communicate.
3. Summary of the chemistry of bioluminescence (fig. 1): Fatty acids within a bacterium are
reduced to aldehydes, then the aldehydes and a second molecule (FMNH2) are oxidized by
luciferase enzyme into a carboxylic acid and FMN, while oxygen is reduced to water. The
luciferase-mediated oxidation liberates energy in the form of visible light.
4. (and 7, oops) An operon is a group of functionally related genes that reside together and are
transcribed as a unit. A plasmid is a circular DNA molecule in bacteria, separate from the main
chromosome. Scientists can use plasmids as vectors as gene-delivery systems for bacteria.
5. The biochemical function of the lux operon is light production. All of the genes for the proteins
that are required for bioluminescence – the three enzymes that manipulate and reduce fatty
acids as well as luciferase – are all part of the lux operon. In addition, the lux operon contains
two regulatory genes: one, lux I, that results in autoinducer, and the other, lux R, that codes for a
protein to which autoinducer binds.
6. In the phenomenon of autoinduction, a diffusible chemical* made by bioluminescent bacteria
diffuses out of the bacteria and – if in large enough quantities – accumulates in the colony and
induces transcription of the lux operons. In other words, when high cell density is detected (quorum
sensing), the lux operon is activated.
*Note: the autoinducer of the lux operon binds to lux R protein, and together that stabilizes RNA
polymerase on the promoter, facilitating transcription.
A single bacterium cannot glow because its autoinducer wouldn’t diffuse back in. Because light
production requires a tremendous amount of energy, free-living bacteria in the ocean do not waste this
energy expenditure that probably offers them no benefit.
8. Cutting enzymes, called restriction endonucleases, digest DNA in a specific way, leaving “sticky
ends” that facilitate splicing. DNA ligase is the enzyme that pastes DNA together. The result is
recombinant DNA!
9. The process by which bacteria scavenge DNA from their environment is transformation.
10. Competent cells are cells that have been chemically modified to become “leaky,” allowing
uptake of DNA.
11. To make E. coli competent, we will suspend them in cold calcium chloride. The Ca2+ ions
stabilize transient pores in the cell wall/membranes.
12. The name of the recombinant plasmid with which we’ll transform E. coli is pUWL 500. “UWL”
stands for University of Wisconsin, La Crosse.
IB Biology Year 1
13. The two sources of DNA “cut and pasted together” to make pUWL 500 were
 pGEM plasmid DNA, sold by a biotech company (Promega), &
 genomic DNA from Vibrio fischeri (i.e., the lux operon)
Both the plasmid and the V. fischeri DNA were cut with a restriction enzyme called Sal I. Sal I was the
first restriction enzyme isolated from Streptomyces albus bacteria.
14. The gene to resist ampicillin was included in our recombinant plasmid for screening. In other
words, only transformed bacteria would possess the ampicillin resistance gene enabling them
to live in the midst of agar with ampicillin.
15. DH5-alpha is classified in Biosafety Level 1, the level with the least risk . According to
Wikipedia, BSL1 is for “… work involving well-characterized agents not known to cause disease
in healthy adult humans, and of minimal potential hazard to lab personnel and the
environment.” My MolecuLab Teacher’s Manual states that DH5-a doesn’t make restriction enzymes,
cannot “have sex with” other bacteria, and is a fragile strain that needs rich agar to grow, decreasing
the likelihood of survival outside the lab.
BSL1 Precautions include
 wearing gloves and some sort of facial protection
 using separate waste receptacles for contaminated materials
 washing one’s hands with anti-bacterial soap
 washing all exposed surfaces of the lab with disinfectants
 being supervised by someone with general training in microbiology