Lab Five: Gene Fusions

What is the goal of this lab?

Observe changes in expression of your gene of interest under different environmental conditions and understand GOI response to environmental stress.

What is insertion in Gene editing?

Insertion in gene editing is the process of adding new DNA sequences to a genome to introduce new traits or functions. This technique can enhance gene expression or modify the organism's characteristics.

What is transformation in cell biology?

The uptake of DNA by the cell.

What is the flow for transformation in the yeast cells in lab?

Live Yeast Cells + DNA Fragments → Fragment Integration → Transformed Cell

What is the transformation mix in this lab?

PEG 3350 (50% e/v), LiAc (1M), and Single Stranded Carrier DNA (2 mg/mL)

What i sthe protocol for Transformation?

Two samples both get yeast cells and transformation mix, but only gets PCR product. This makes the other a negative control. (Grown on SDS-HIS plate)

What is the first step in designing a hypothesis in this lab?

Find a related gene based on functional association data

What is the second step in designing a hypothesis in this lab?

Analyze Protein Localization

What is the third step in designing a hypothesis in this lab?

Determine Expression Levels on gene.

What are we doing in Lab Six?

Test Conditions with Spot Dilution

What are we doing in Lab Seven?

Read out: Cell concentration andq GFP expression

There are multiple different types of stresses. What is osmotic pressure in this lab?

0, 0.5M, 1.0M NaCl concentration

There are multiple different types of stresses. What is oxidative pressure in this lab?

0, 2.0, 5.0, 7.0 mM hydrogen peroxide

There are multiple different types of stresses. What is metal toxicity in this lab?

0, 5.0, 10.0, 15.0 mM CuSO₄ concentration

There are multiple different types of stresses. What is ethanol treatment in this lab?

0%, 10%, 20%, 40% ethanol

There are multiple different types of stresses. What is temperature stress in this lab?

cold (shift from 30ºC to 20-25ºC) and heat (50ºC, 30 minutes) shock

There are multiple different types of stresses. What is dna sensitivity in this lab?

U-V light exposure immediately after spots dry, expose to UV light (agar down) for 0-1 minutes. Wrap for one minute for incubation to prevent light dependent repair.

There are multiple different types of stresses. What is an alternative carbon source in this lab?

Instead of glucose in a YPD, 3% glycerol or 3%$ galactose can be used

What is Gene Mania?

A software used to predict the function of unknown genes by using association data of input genes and measuring genetic interactions,. protein interactions, pathways, co-expression, co-localization, and protein domain similarity.

If the members of a gene list make a complex, then…

Gene Mania can predict posibilities

What is a physical interaction?

When two gene products are linked if they were found to interact in a protein-protein interaction. Info found in protein interaction data bases, BioGrad and Pathway Commons

What is a Genetic Interation?

When two genes functionally associated if the effects of perturbing one gene were found to be modified by perturbation to as the other gene. This relationship can indicate how genes cooperate or influence each other's function.

What is co-expression?

When two genes are linked if they have the same domain.

What is the shared protein domain?

Two genes are linked if they have the sam domain.

What is co-localization?

Two genes are linked if both express the same tissue or gene product 

What is a pathway?

When two gene products are linked if they participate in the same reaction within a pathway.

What is SPELL?

Also known as Serial Pattern of Expression Data sets, which is a query driven search engine for large expression microassay that identifies most information datasets to those that are not.

What is the yeast protein localization database provide?

Information about sub-cellular localizations of proteins in yeast for studies YPL was used, GFP fusion and Confocal Laser Scanning page.