144 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Fundamentals
1. What is the equivalent of a promoter in RNA?
2. Describe why a ribosome is a hybrid structure
3. What is the difference between RNA transcript and rRNA?
4. What is the difference between the codon and the anticodon?
5. How does the process of translation use the Four B’s?
6. What is fMet?
7. What are the three different “kinds” of RNA you have learned about? Explain the differences.
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145Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Check Point!
You’ve accomplished quite a lot on your journey so far, congratulations! Below is a 19 point checklist which
summarizes what we learned and did in Chapters 1-5. Review it before going further, and make sure to repeat
any exercises or breakouts if you feel it necessary!
LB agar plates are made. They are the food (sugar, amino acids) and substrate (solid agar scaffolding in a
petri dish) that E. coli can grow on. Non-selective LB agar plates are used to grow “Blank cells”, while selective
LB agar plates are used to grow genetically engineered cells that have a selection marker.
Blank cells are collected from a stab, streaked onto a non-selective LB agar plate and incubated at 37 ˚C
for 12-24 hours so that fresh, fast-growing, non-engineered individual colonies can be used for the genetic
engineering experiment.
Chemically competent cells are made in a cold environment by collecting fast-growing E. coli cells and mixing
them into Transformation Buffer. Transformation Buffer is a liquid that contains salts such as calcium chlo-
ride so that the cells are better able to take in DNA.
A DNA plasmid is mixed into the chemically competent cells, incubated and then heat shocked at 42 ˚C to
help the DNA plasmids pass through the cell membrane and cross into the cells. The cells are cooled to trap
the DNA inside.
The cells are recovered in Recovery Media at 37˚C to begin their growth cycle, division, and so that they
start expressing their selection marker, such as an antibiotic resistance enzyme.
To express the selection marker, the DNA plasmid includes a specic gene. This gene sequence has
a promoter, RBS, and protein coding sequence (with a start and stop codon). After transcription and
translation, the gene results in the expression of a protein enzyme called chloramphenicol acetyltrans-
ferase that can break down the antibiotic:
Sigma factors bind to the promoter and recruit the RNA polymerase which creates an initiation
sequence. It then tries to escape the promoter
If the RNA polymerase escapes the promoter, it moves downstream on the DNA molecule,
unzipping it and, as the correct complementary ribonucleotide enters the polymerase, adds it
to the string of RNA
Rho-dependent (Rho proteins) or rho-independent factors (poly-Ts and/or terminator hairpins)
cause the RNA polymerase to “slip offthe DNA. The RNA polymerase then releases the RNA
transcript into the cytoplasm of the E. coli cell - transcription ends.
Initiation factors bumping around the cell bump into the RBS sequence of the RNA transcript
and the ribosome which also binds to the RBS and locks in thanks to ribosomal RNA (rRNA).
If a start codon is present, a special starter tRNA (fMet) which is bound to the initiation factors,
binds to the ribosome and kicks starts translation. The ribosome rides down the RNA tran-
script, and many aminoacyl-tRNAs that are complementary to the particular RNA codon in
the RNA transcript allow the ribosome to know what amino acids to add to the growing amino
acid string.
The ribosome encounters a Stop Codon where a release factor resides and concludes the trans
-
lation of the amino acid string. The ribosome falls off the RNA transcript
While translation is happening, as well as afterward, the amino acid string folds upon itself to
form a three-dimensional macromolecule called a protein. It is the three-dimensional struc-
ture of the protein that determines its function.
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146 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
After 24-72 hours, colonies of bacteria can be seen on the selective experimental LB agar plates. The colo-
nies on the plate will be expressing the trait that they were engineered to show, such as a change in color
due to the creation of a colored protein.
Just like a sample of bacteria was collected from a stab, some bacteria from colonies on the plate were
collected and used to culture, or amplify bacteria on further selective LB agar plates.
Cells are incubated for 24-48 hours until they sufciently expressed their trait (such as color). During this
time, the cells were completing transcription and translation, which resulted in the trait.
Cells packed full of your colored proteins are collected and added to a Lysis Buffer which contained a surfac-
tant called Triton X-100, and a Lysis Accelerator called HEWL which was an enzyme that actively degraded
the peptidoglycan in the intermembrane space. The samples are incubated so that the cells lysed, and the
colored proteins were released.
The lysed cells are microcentrifuged at high speeds (more than 10,000 x g) so that all the cell debris is
pelleted at the bottom. The sample’s supernatant is poured into a syringe with a microlter attached.
The sample is sterilized by passing it through a 0.22 um pore diameter lter and stored in a tube for later
use.
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