BISC411

                                    EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL

                                                            Induction Protocol

In late afternoon of the previous day, instructor inoculates 5 ml of LB containing 100 ug/ml ampicillin with T4 expressing bacterial cells (from a growth plate colony preferably). Grows it overnight, shaking at 300 rpm at 37oC.

Approximately 3 hours before the start of the lab (around 9:30), instructor seeds 25 ml of LB containing 100 mg/ml ampicillin with 0.25 ml of the overnight culture. Shakes this at 300 rpm for 3 hours. (To OD 595 of .80-1)

Instructor makes fresh IPTG stock just before needed. Adds 30 mg to 1.5 ml LB.

Students then remove 3 ml of the bacterial culture and put aside, labeled as uninduced sample. Shaker temperature is lowered to 30oC.

Students add 275 microliters of the IPTG stock to the 22 ml culture remaining. (This is a total of .25 mg/ml IPTG). Cultures are shaken at 150 rpm from now on.

Every 30 minutes, students remove a 3 ml aliquot of induced culture for analysis. Aliquots are taken at 30 minutes, 1 hour, 1 hour 30 minutes, 2 hours, and 2 hours 30 minutes.

Each 3 ml aliquot is sonicated on setting 8 with three 20 second bursts and 20 seconds of rest between bursts. Following sonication, students split the sample into 2 microfuge tubes and spin out the debris for 2 minutes in the microfuge.

As soon as you have the clarified lysate you can proceed to assay that time point as described below.

                                                    Lysozyme Activity Protocol

Prior to the start of the lab, the instructor obtains from the prep room a culture of micococcus luteus that has been grown by the staff for about 3 days. Culture can be stored in frig until needed. Instructor dilutes the culture 1:1 in phosphate buffer and measures the OD 450. OD should read between 0.5-0.6. (Students should check this out before they use it for their assays. Adjust accordingly with phosphate buffer).

Lysozyme activity is measured at OD 450 by tracking decrease in optical density as the substrate’s cell wall is broken by the enzyme if it is present.

While waiting to collect the first sample, students should use their previously made lysozyme stock solution (the one you determined should give ideal results) and do a lysozyme assay with it as you did before. This will tell us that the microccus luteus substate is suitable for the experiment. You should obtain a result similar to the earlier result.

Students can then assay 1 ml of their time samples. First, the spectrophotometer is blanked with water to read zero OD. Then a cuvette tube is filled with 2.5 ml substrate.(NOTE: WE USE DIFFERENT VOLUMES TO MEASURE ACTIVITY OF OUR BIOLOGICAL SAMPLES THAN WE USED BEFORE. WHY DO YOU THINK WE NEED TO DO THIS?) To this they add 1 ml of sample, immediately vortex, and immediately put into spec. Readings are taken every 15 seconds for 3 minutes. Note: have paper and pencil ready to take these readings and a watch with a second hand to measure the time elapsed.

To generate samples for future polyacrylamide gel electrophoresis, add 50 microliters of the lysate to 50 microliters of the 2x loading buffer. Vortex. Store at -20 degrees C.

Put all remaining sample in freezer also to be used for protein concentration measurement next time.

For the lab report, you will need to calculate the total protein concentration of each time point and the units of lysozyme activity per ml of induced culture for each time point. See the instructions for this, linked to our course website.