SARS-Cov-2
How do you detect the presence of viral infection?
look for viral ssRNA genetic material
How is GFP structured?
cylindrical structure formed from tightly packed beta pleated sheets
True/False: Denaturing the proteins will remove their fluorescence.
True
To denature the protein samples, add 25μl of protein denaturing solution (F) to each of the tubes labeled "gfp denatured" and "bfp denatured". The denaturing solution contains sodium dodecylsulfate (SDS) and 2-mercaptoethanol.
What's a promoter?
landing site for RNA polymerase
What is ELISA?
allows us to detect soluble substances (antibodies)
What happens in the reverse transcription step in the RT-qPCR test?
convert viral RNA to a double stranded DNA because we need the DNA template for PCR to work
How is a chromosphere made?
chromophore is formed from the side chains of three amino acids as the protein folds: Serine, Tyrosine, and Glycine. The side chains of Ser65 (number refers to the where the amino acid occurs in the polypeptide chain), Tyr66, and Gly67 react and link via covalent bonds between the side chains to form the chromophore.
What's SDS?
SDS is a strong detergent and crucial reagent in SDS sample buffer that is used in protein gel electrophoresis, or SDS-PAGE.
What's IPTG?
arabinose tetracycline
used to treat infections caused by bacteria
What is it used for?
Looks for antibodies that were made by someone when they were infected by a virus
Lab = HIV
True/False: SARS-Cov-2 contains a phospholipid bilayer.
true
How do you separate GFP from the other stuff considering cells have thousands of proteins, DNA, RNA, etc?
First, we will discard any material that is not water soluble. We will then separate the GFP from the water-soluble material using chromatography that takes advantage of its hydrophobic properties
How do we confirm the purification of gfp and bfp?
The purification of gfp and bfp will be confirmed by examining the molecular weights of the proteins, using denatured SDS polyacrylamide gel electrophoresis.
IV and DV?
IV: Bacteria introduced to plasmid
DV: Bacterial growth on plates and UV glow
True/False: If the patient is negative for HIV, there will be a primary antibody.
False. If the patient is negative for HIV, there will be no primary antibody to bind to the antigen and in turn, no secondary antibody binding.
What can the reverse transcriptase do?
Destroys the DNA in the RNA/DNA hybrid molecule
Makes complementary DNA to an RNA template
Synthesizes complementary DNA strand to a DNA template
In the gel-filtration chromatography, proteins are separated by what? How does the separation process occur?
proteins are separated according to their size
When a sample is passed through a column packed with a matrix of porous beads, low molecular weight proteins flow through and around the beads in the direction of solvent flow, and high molecular weight proteins flow around the beads without interacting with the matrix material. Consequently, progression through the column is slower for smaller proteins than for larger ones.
Can the gfp native fluoresce with a short or long UV light?
The protein in its native form can be shown to fluoresce with a long wave U.V. light.
What were the results of the cells on LB + amp + IPTG plate?
IPTG was able to change the shape of the lac repressor. This allowed E. coli RNA polymerase to code the T7 RNA polymerase gene. RNA polymerase recognizes T7 and binds which leads for GFP to be coded and produced.
What's the purpose to the protein containing buffer in Step 3?
prevents non-specific interactions between the antibody and the plastic wells
True/False: A sample containing viral RNA will get dimmer over time.
False
A sample with viral RNA will get brighter. If the fluorescence crosses a certain threshold, set above expected background levels, the test is positive. In a sample from an uninfected individual — where no viral material exists in the first place — no copies will be made, and no light will be emitted. The fluorescence threshold isn’t reached — the test is negative.
When collecting column fractions of GFP proteins, we have to elute the column with elution buffer. Why do we add the buffer slowly?
Name 5 things that are needed in PAGE in addition to the protein sample.
What's Amp r gene and GRP gene?
GRP gene gives instructions to make green fluorescent protein
Amp r gene that encodes for B-lactamase (enzyme that contains the antibiotic, ampicillin, so the antibiotic can be deactivated)
What's the IV and DV?
IV: samples of blood/serum/saliva
DV: antibodies connected to virus being tested