Assignment:
Recombinant protein expression controlled by lac operator
PROTEIN EXPRESSION
Underlying molecular principles:
Explain the genetic components of the lac operon regulating the IPTG-inducible expression of T7 RNA polymerase in the TrcHisB/T7 and DH5 system
Hands-On Skills:
1. Inoculate and grow a transformant DH5 cell line
2. Assess the cell growth phase by absorbance at 600nm
3. Induce protein expression by adding IPTG
4. Harvest cells from a liquid culture
5. Prepare one buffer solution to be used in a future laboratory class
1. Perform a quick review of literature on recombinant protein expression in E. coli and indicate the protein yield reported in two research articles of your choice. Make sure you specify the units for the protein yield. For each article describe the nature of the protein that was overexpressed (membrane protein, cytosolic protein, ...). Estimate how much of your recombinant T7 RNA polymerase enzyme you might expect from 50ml of liquid culture by assuming a yield comparable to the ones reported in the two articles you selected. A hard copy of each of your two articles along with your answer should be handed in to your TA when entering the lab
2. The overview figure provided in Experiment A shows the planning for the control aliquots to be assessed by SDS-PAGE in lab 6. Due to space limitation in the shaking incubator, some controls will be done collectively. Explain the information to be obtained from each of the two extra sets of controls to be done collectively:
a. The inoculated 50ml culture containing 200ug/ml ampicillin is to be divided in two fractions of 25ml. At time zero of the induction, the first 25ml fraction is to be induced with 1mM IPTG whereas only water is to be added to the second 25ml fraction. A 1ml aliquot is to be withdrawn from each of the two fractions at both time zero and after a 1.5 hr incubation. ( /2.5 MARKS)
b. A liquid culture of non transformed DH5 cells will be incubated without ampicillin, and a 1ml aliquot is to be collected at time zero and the very end of the induction treatment with IPTG.
Attachment:- Laboratory Class.rar