Measurement of -galactosidase activity with different substrates (week 6) As des
ID: 150579 • Letter: M
Question
Measurement of -galactosidase activity with different substrates (week 6)
As described last week, our goal for this set of experiments is to quantify differences in the induction of -galactosidase under a variety of conditions, to better understand how promoter elements IN COMBINATION affect gene expression.
The goal of today’s lab is to compare how the promoter responds to inducers (to activate gene expression) and repressors (to prevent gene expression). As a follow-up to last week, our first set of experiments will test how well IPTG induces gene expression. Our second set of experiments will determine what happens to expression if glucose also is present in the culture. Would bacteria want to go to all the trouble of making these proteins if other sugars are freely available?
lacZ+ cultures were started at 37°C prior to lab and should be growing in the exponential phase at the start of the experiment. We will split the culture to 6 separate flasks, and you will add the following reagents:
Cultures
nothing (control)
1 M IPTG
10 M IPTG
100 M IPTG
10mM Glucose
100 M IPTG + 10mM glucose
Note: your lab bench will get one of the 6 cultures above and will be responsible for adding IPTG and/or glucose. However, you and your lab partner will do assays on all 6 cultures.
Return the cultures to the 37°C incubator. After 60 minutes of incubation (when the OD595 is ~0.4-5), you will remove a 2ml sample from each culture flask. Measure the activity of each culture using our standard protocol.
Calculate how much IPTG and/or glucose you will add to 50 ml cultures.
No additions
______ l of 1mM IPTG stock
______ l of 1mM IPTG stock
______ l of 100mM IPTG stock
______ l of 1M glucose stock
______ l of 100mM IPTG stock and _______ l of 1M glucose stock
Explanation / Answer
1) .05
2)0.005
3).005
4).005
5).005 and .005
all calculations are done usinge simple M1V1=M2V2