155Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Step 4. Add cell extract beta-galactosidase to the substrate
Get ready for chemical wizardry! Using a pipet, add a small amount (50-100 uL) of cell extract to each substrate
tube. The plastic pipet will have a small 100 uL marker on it to help guide you. Place the lids on rmly and
invert them to mix them thoroughly. Place the tubes on your DNA Playground Hot Station set to 37 ˚C and sit
back and let the beta-galactosidase do its job! Over the course of the next 24 hours, the samples will become
increasingly blue and yellow. You’ll see yellow start to appear in minutes (usually within 10 minutes), and the
blue will appear over the course of hours.
Enzymatic Processing Going Deeper 6-2
You have now completed enzymatic processing! In both exercises, you engineered the cells to produce
protein enzymes (Atf1 & beta-galactosidase) that were able to complete the Four B’s and ultimately interact
with substrate molecules (isoamyl alcohol + acetyl-CoA or Xgal+H
O) and turn them into products. But why
did you do the Smell-it Kit within the cells vs. the Blue-it Kit as an extract? These are both useful methods
and choosing between them depends on the many factors such as the stability of the protein enzymes, the
availability of the substrates in the cell, and the cost of buying substrates.
In Exercise 1, a key reason for using streaked cells on plates, rather than an extract, was that one of the
two substrates, acetyl-CoA, is very expensive to purchase and you needed the cell to keep microfacturing
it naturally for your chemical reaction. Because of this, lysing the cells would have stopped the cells from
making more acetyl-CoA, and you would have had only a small amount of acetyl-CoA for the reaction (when
you lyse the cells, they will not create any more acetyl-CoA). Doing the reaction in living cells means the
cells continue to grow and they can keep producing acetyl-CoA, and so long as you have lots of IA available,
the reaction can continue to occur. This means you could keep adding IA to the cells and they would keep
processing it into isoamyl acetate! For reference, 0.1 grams of acetyl-CoA costs more than $1000 to buy!
Including it in the kit would sure increase the cost of the Smell-It Kit! Let the cells make it for you!
In Exercise 2, only one substrate molecule is needed for the chemical reaction, and that chemical is reason-
ably priced. There is no need to have the cells continue growing to create a substrate. Because a single
beta-galactosidase enzyme can cause the chemical reaction to happen thousands to millions of times
during its lifetime, you can simply use the extract with the engineered enzyme and add the X-gal substrate.
An alternative protocol for Exercise 2 could involve growing cells in a petri dish, similar to Exercise 1. Pour-
ing or pipetting a small amount of the X-gal substrate over the top the plate of cells, or adding it into the
LB agar when you pour the plates is common. The substrates in the LB agar can cross into the cells. The
enzymes inside the cells will begin catalyzing the same chemical reaction inside of the cells - the colonies
of bacteria would turn from white to colored.
Naming Enzymes Pro-tip
Protein enzymes are usually easy to spot because their name will end in “…ase”. Generally, the beginning
prex of the enzyme name will give you some historical or chemical context about what the enzyme does,
and then “ase” sufx is added to the end.
In Exercise 1, alcohol acetyltransferase (Atf1) is a great example. One might deduce from its name that
it binds alcohols and transfers an acetyl group to it. In Exercise 2, the enzyme is beta-galactosidase. One
might deduce that it can catalyze reactions involving beta-galactoside sugars. And if we think even further
back to Chapter 4, RNA polymerase was the enzyme that connects ribonucleotides into a polymer.
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