Gene expression
Instructions:, Transgenic Fly Virtual Lab,and Lactose Lab handouts to help for this assignment . You will be submitting your answers to SafeAssign, so please use your own words.
The document you submit to Safe Assign should only have the question numbers and your answers in it. Please delete the instructions, video links and questions themselves so that they do not show up as matches to other classmates’ assignments in SafeAssign.
You want to look at how certain changes in LCT promoter sequences could affect LCT expression. You decide to make a reporter construct/plasmid in order to do this.After putting your plasmid into bacteria to make lots of copies, you will eventually put your plasmid into human cells in cell culture (not into Drosophila).
- Add and label at least three types of sequences/genes you would want to include in the reporter plasmid. In a paragraph below, please describe the purpose of each of these three sequences.
- While bacterial cells are not necessarily the best cell type to use to look at human gene expression, you want to put your plasmid into the bacteria to make extra copies. Name one method you would use to get your plasmid into bacterial cells. Then describe the general steps of that method
- You eventually transfect your plasmid into human cells in cell culture. Let’s say you have three different constructs:
- Control promoter
- Promoter with a deletion mutation in a enhancer binding site
- Promoter with a deletion mutation in a repressor binding site
You transfect each into different cells and measure the reporter level in each. Create (in Excel)and insert (as a pdf or jpg image) a graph of what you would expect to see from each cell type. You should label the axes (with units), but do not need to worry about exact values (you can make up ones that seem OK).
- Why is using a reporter advantageous when trying to measure gene expression?
- You identify a family whose babies have severe lactose intolerance. You want to identify why this is the case. You suspect there might be altered LCT expression in this family. Come up with two possible sites for a mutation in this family, and explain how those mutations might cause the symptoms.
- Now explain why (or why not) you could use exome sequencing to detect each of the two mutations you described in question 5.
