Problem Set 5

9) What does it mean for a insert sequence to be "in frame" with the vector sequence? When would it not be necessary to make the insert sequence in frame with the vector?

A DNA coding seqence has the genetic code that is necessary to make a certain amino acid sequence through the processes of transcription and translation. The codons of the genetic code, which contain three nucleotides each and code for the individual amino acids, are formed from a particular reading frame that starts with the ATG (start codon). If a single nucleotide is added to the coding sequence, this is called a "frameshift mutation" because now there is a different grouping of codons, which will result in a different amino acid sequence. If the ATG is on the plasmid and upstream from the first restriction enzyme site (or 5' site), the sequences after the cut site must have the same reading frame. This requires careful placement of the restriction enzyme sequence upstream of the coding sequence (of the cloned gene) in the insert DNA. If the start codon (ATG) is downstream from the first restricition enzyme site (in other words, it is included inside the inserted sequence and does not use an ATG or other plasmid sequences), then it does not have to have the same reading frame.

5) How are the derived fluorescent proteins EGFP, ECFP and EYFP different compared to the parent GFP protein?

EGFP (enhanced GFP) has two point mutations. One increased the folding efficiency of the protein and enabled it to be more stable at normal room temperature (whereas the native protein preferred colder, marine temperatures). The other mutation changed te excitation peak to 488nM, making it a better FRET acceptor.

4) Describe how fluorescent proteins can be used as tools in research.

Fluorescent proteins are commonly used as molecular tags in protein research because they can be added to the N or C terminus of a protein without (usually) changing its function. They serve as markers for gene expression or cellular localization. They are commonly used to screen drug compounds, evaluate the effectiveness of gene therapy, visualization of biological pesticide use and monitoring genetically altered microbes, just to name a few applied uses.

2) Why did we use the compound imidazole to remove our His-tagged fluorescent proteins from the nickel resin? Why was there a low concentration of imidazole in the lysis/wash buffer that was used to bind your fluorescent proteins to the resin? Explain.

Imidazole and histidine have structural similarities, and imidazole will compete for histidine binding to the Ni-NTA resin and allow for elution of the His-tagged protein. The low amount of imidizole in the lysis/wash buffer will compete with the dual histidine repeats (2x His) that are naturally occurring in the protein's strucuture and prevent their binding because they have the same low binding affinity for the resin. This will ultimately reduce background because the high affinity binding of the 6x His tag will predominate.

10) After plating your transformed bacteria to the agar plate, you check on it next week only to find a "lawn" of bacteria growing on the surface. (A lawn is when the plate is covered in bacteria, and individual colonies can not be identified). What is the most likely explanation for this occurring?

The ampicillin was not added to the plate and it lacked the proper selective marker.

1) In fluorescence, why is the emitted (fluorescent) light always a longer wavelength than the light that excites the fluorophore? Explain each step of the excitation and emission process using diagrams.

The emitted light has lost energy due to heat dissipation, and therefore is a longer wavelength. (See page 185 for excitation/emission diagram during fluorescence).

3) The promoter region of your pRSET vector contains an element called the RBS (see plasmid map for the first exam-level question). What is the function of this element? What is the function of the element labeled T7 gene 10 leader? How does it work in coordination with the RBS?

They both enhnace binding of the transcript to the bacterial ribosome.

8) Sometimes the supercoiled, nicked circle, and linear molecules are called "form I", "form II", and "form III" DNA, respectively. If you were to raise the temperature of form II DNA solution (with a single nick in the DNA molecule) until it was boiling and the strands denatured, what would you find when the DNA was cooled? a) A supercoiled molecule (form I) b) A nicked circle (form II) c) A double stranded linear molecule (form III)

d) One single stranded linear molecule and one single stranded circle

7) (Circle one) A band of DNA would travel faster/slower in a 0.6% agarose gel compared to a 2.0% gel.

faster/