experiment 3: expression of rGFP in E.coli

E.coli

-has hard outer cell wall made of peptidoglycan (formation prevented by ampicillin)

-double membrane system (bilipid membrane inside and outside)

-approximately 4000 genes, ~4000 different proteins

-we're only interested in 1 gene being expressed (transcribed, translated), over express the 1 gene you're interested in (using recombination events)

What is GFP?

-green fluorescent protein found in jellyfish

-wild type: ORF codes for 238 amino acid protein (MW ~ 27 kDa)

-we will use rGFP that has a His6 tag on n-terminus and another tag on c-terminus (MW > 27 kDa)

How/why does GFP "glow" green?

-chromophore "antenna" protected inside beta barrel, stable to changes in oxidation/reduction chemical reagents, pH, temperature

-3 amino acid sequence: Ser65, Tyr66, Gly67

-cyclization reaction between Ser65 and Gly 67, pushed Tyr66 out of peptide backbone and forms chromophore (antenna) that likes to pick up energy

-picks up energy at one wavelength, loses some energy as heat, vibrational, or rotational energy, remainder of energy is fluoresced/radiated out at longer wavelength with less energy

-λex: 395 nm (blue), λem: 510 nm (green)

-mutation within/around allows you to alter excitation and emission wavelengths, lead to red, blue, etc

dual labeling

-amino acid substitution mutants result in altered excitation/emission spectra

-have GFP and RFP present

-they colocalize and overlap, create yellow fluorescence

create fusion proteins

-to visually identify/locate "your favorite protein" in vivo

-perform inter/intra cellular traffic studies

create promoter fusions

-for in vivo expression studies

-what conditions/inputs control the promoter? (try input A, do we get fluorescence? try input B, do we get fluorescence? if yes, then the input controls the promoter)

-does a particular condition/input control different promoters? (promoter 1 in front of GFP, promoter 2 in front BFP, try input C and see if GFP or BFP is seen)

Why was the GFPuv gene placed into the pRSETA plasmid?

-the pRSETA plasmid has many features that allow for easier expression of rGFP. These features include the His6 tag, the Xpress epitope tag, and the inducible T7 promoter. This plasmid also contains an Amp gene, which helps maintain selection for this plasmid in an environment containing ampicillin. This acts as a selective marker, since any plasmids without the Amp resistance gene will be destroyed

How are a "calculated MW" and an "estimated MW" of a protein determined?

-Calculated MW: first determine the exact number of specific amino acids within the protein (example: 28 Met, 42 His, 19 Thr, etc). Multiply each number of specific amino acids by the specific MW of that amino acid. Add up all of the masses to get the total mass for the whole protein (Done this way in Prasher paper in regards to GFP gene)

-Estimated MW (2 Methods)

1) SDS-page electrophoresis (Lab 6): get relative molecular weight, since you compare location of protein with DNA ladder with pre-determined mass

2) Know total number of amino acids present in the protein. Multiply this number by 120, the average MW of an amino acid

Why was the pRSETA -GFPuv transformed into the E.coli strain BL21pLysS?

There is a lot of information known about this particular strain, which makes it easier to make informed conclusions when using it for gene transformations. For example, when using this strain, several conclusions can be made about the procedure used. These include

-Having the gene of interest

-Selectable plasmid

-Inducible with T7 promoter

-Can overexpress specific genes

What is the general process of inducing expression of rGFP?

rGFP is induced by adding the compound IPTG to the bacteria. This compound inhibits the Lacrepressor protein. As a result, the pLac promoter is allowed to generate a larger amount of the T7 RNA Polymerase protein. This protein binds to the T7 promoter, which leads to the production and expression of rGFP

Why was the GFP placed under the control of a T7 promoter?

-T7 is an inducible promoter (induced by IPTG). Placing GFP under control of the T7 promoter allows for easier control of GFP expression.

-note: only genes downstream from T7 promoter get expressed

What is the purpose of adding IPTG to the growing culture?

-The phase of maximal growth is the log phase. During this phase, the bacterial cells are the most abundant and the healthiest. During this time, induction with IPTG allows for the maximum GFP expression. Additionally, since the overexpression of GFP accompanying induction is energy intensive, it's very important for the cells to be at their healthiest state. (Note: the cells will die shortly afterwards due to the overexpression of the protein.)

-O.D of 0.1 → O.D of 0.5 represents 5-fold growth in number of bacterial cells.

What was the purpose for the pLysS plasmid?

The pLysS plasmid encodes for the lysozyme protein. This protein can degrade cell walls from the outside to the inside. This is a preferential process that makes the freeze-thaw method easier to lys cells

What is the purpose of the adding ampicillin and chloramphenicol to the media?

Having ampicillin and chloramphenicol allows for plasmid selection. Only the plasmids containing the genes for Amp and Cam resistance are able to survive the Amp and Cam antibiotics present in the media. Additionally, their presence in the media prevents contamination from other bacteria. This is because the antibiotics interfere with cell wall formation and translation of the bacteria without plasmids containing the resistance genes. In the end, the Amp and Cam antibiotics kill bacterial cells, but not resistant plasmids.

Why do we take culture samples before and after the addition of IPTG?

Taking culture samples before and after the addition of IPTG shows what happens before (G0) and after (G3) adding the inducer. Doing this shows that the bacteria doesn't fluoresce initially. This allows for a comparison to the G0 baseline

Who first discovered GFP?

-in 1962, Shimomura reported the the purification and some of the properties of aequorin from medusa of Aequorea victoria, also reported existence of GFP

Who first cloned GFP?

-in 1992, Prasher cloned the gene for GFP

What was the first transgenic organism to express GFP?

-in 1994, Chalfie reported expression of GFP in E.coli (single cell prokaryote)

What was the first transgenic eukaryote to express GFP?

-in 1994, Chalfie reported expression of GFP in C.elegans (multi-cellular eukaryote, nematode)

negative control

-shows what negative result will look like in an assay; done usually by leaving out a substrate/enzyme

-can't use water b/c best control has everything you want EXCEPT the one variable that you're testing (using water would remove multiple variables, which would mean that whatever results seen with water aren't true controls)

-controls are CONTEXT DRIVEN; harder to assign pos and negative controls in bacterial cultures (easier to classify in enzyme assays) - G0 considered a control, but not "negative" or "positive;" just acts as baseline

positive control

-used to show what positive control looks like

-used to how that assay works

-harder, since must use something that you know has all of the substrates/enzymes that are being tested

correct way to draw plasmid/plasmid map

-Line in form of circle; represents double stranded DNA

-Genes represented as boxes/rectangles

-Indicate promoter that controls specific genes