Lab Four: Designing hypotheses and GFP Fusions

What is the first objective of Lab Four?

To understand how protein sequences and unction are related.

What is the second objective of Lab Four?

To know the role of sequence alignment techniques in identifying protein function.

What is the third objective of Lab Four?

To understand the role of protein superfamilies in classifying proteins.

What is special about S. cerevisiae?

It was the first eukaryotic organism to have its entire organism genome sequenced, providing insights into eukaryotic biology.

What is a homolog?

A sequence that shares a common ancestor with another; a gene related to a second gene by descent from common ancestral DNA sequence.

What is an Ortholog?

A gene in different species that evolved from a common ancestral gene by speciation, often retaining similar functions.

What is a Paralog?

A gene that results from duplication within a genome, which may evolve new functions.

What is a Domain?

A distinct modular region of a protein that serves a particular function, such as DNA bonding.

What is a Family?

A group consisting of proteins that are more than 25% identical in amino acid sequence across their entire length, shares structural function and other related functions.

What is a SuperFamiliy/Clan?

A group of protein families that are related by detectable levels of sequence similarity reflective of an ancient evolutionary relationship

What is BLAST?

A bioinformatics tool used for comparing primary biological sequence information, such as the amino acid sequences of proteins or the nucleotides of DNA.

What type of alignment is used in the BLAST Program?

A pairwise alignment method used to compare sequences.

What is the E-Value in a BLAST alignment?

A statistical measure that indicates the number of hits one can expect to see by chance when searching a database of a particular size. A lower E-value suggests a more significant match.

What is gene fusion?

The process of introducing a gene of interest into a plasmid that contains a GFP reporter gene.

How do you tag genes of interest?

By using a green fluorescent protein (GFP) and attach a detectable sequence to the target gene to visualize expression.

What is Green Fluorescent Protein?

A protein that glows with a bright green fluorescent under UV light, commonly used as a marker in biological research to visualize gene expression and protein localization. First found in marine jellyfish Aequorea victoria.

What is the goal of using GFP?

Understanding how gene expression changes in response to environmental stress.

What does capital mean in a gene for example “HIS”?

It means a dominant gene , in this case a dominant histidine biosynthetic gene. The opposite is for a recessive gene use lower case.

When looking a how to name certain organisms such as the following: YNL323w. What does the “Y” mean?

represents the yeast genus, indicating that the organism belongs to the species Saccharomyces cerevisiae, a type of yeast commonly used in research.

When looking a how to name certain organisms such as the following: YNL323w. What does the “N” mean?

indicates a specific chromosome within the Saccharomyces cerevisiae genome, specifically chromosome number 14 (N).

When looking a how to name certain organisms such as the following: YNL323w. What does the “L” mean?

It indicates which chromosome the gene is located on. In this case the Left One.

When looking a how to name certain organisms such as the following: YNL323w. What does the “323” mean?

It designates the specific gene identification number assigned to a gene on the chromosome. In this case, gene number 323 on chromosome 14.

When looking a how to name certain organisms such as the following: YNL323w. What does the “w” mean?

It signifies that the gene encodes a protein and is located on the Watson strand of the DNA.

What is PCR used for?

To amplify the specific segment of DNA deigned ffor the Gene of Interest.

What is PCR?

The technique that allows for specific amplification of many copies of DNA molecules from a template sequence.

What are the four thing s required for PCR?

• Template DNA

• Primers

• Mg²⁺ ions

• dNTPs

What are the primers for PCR for?

They provide specificity for the target sequence to be amplified from the template DNA and in solution is both forward and reverse primers.

What is the Plasmid Map?

A graphical representation of a plasmid's structure, showing the locations of genes, restriction sites, and other important elements.

What does GFP encode for in the Plasmid Map?

Green Fluorescent Protein, which emits fluorescence when exposed to UV light.

What does HIS3 encode for in the Plasmid Map?

A histidine biosynthesis gene that allows for the selection of transformed yeast cells that can synthesize histidine.

What does ori encode for in the Plasmid Map?

a functional origin of replication necessary for plasmid replication within a host cell.

What does AmpR encode for in the Plasmid Map?

Ampicillin resistance, allowing for selection of antibiotic-resistant cells.

In a plasmid map are the forward and reverse positions of the primers noted?

Yes, they are indicated by arrows showing the direction of transcription.

What does a forward primer target?

The frist 18 bp of the GFP gene

What does a reverse primer target?

The last 18 bp of the HIS3 gene..

What plasmid will PCR amplify GFP and HIS3 from?

pFA6a

How much of an extension does the forward primer of a plasmid make?

A 60bp extension immediately upstream the GOI start codon.

How much of an extension does the backward primer of a plasmid make?

A 60bp extension immediately downstream the GOI stop codon.

The base pair extensions of the primer are known as the flanking regions of the gene. What will they be recognized by?

The homologous recombindation machinery and recombined with cooresponding sequences in the yeast genome, resulting in a GOI-GFP gene fusion.

What is important about the natural STOP codon of the gene of interest?

That is has been removed form the GOI so that the GFP protein will be fused to your protein of interest.