Index CourseInfo LogIn Syllabus Protocols Other Resources

Index of Project 2 Reports for Spring 2003

Group 1

Kelsey Grant and Vishnukant Joshi

The goal of this project was to express b-galactosidase in COS-7 cells. The Invitrogen Staining Kit procedure was used and it was found that 27% of the total COS-7 cells were transformed by the recombinant plasmid and expressed b-galactosidase.

Group 2

Angela Baioni and Ramadevi Bonthu

The goal of this project was to express b-galactosidase in E.coli cells and to compare levels of expression in IPTG induced cells to the level of expression in uninduced cells.

Group 5

William Bush and Britany Sutherland

The goal of this project was to determine the time that it takes to reach a maximal level of b-galactosidase production with the pcDNA3.1His B/Lac Z/SD plasmid in IPTG induced JM109DE3 cells. It was found that maximal protein production is reached in three hours. This is the same level of b-galactosidase activity that cells reach when they are induced overnight.

Group 6

Wesley Smith and Nicole Yeager

The goal of this project was to purify and compare the activity of b-galactosidase isolated from pcDNA3.1His B/Lac Z/SD transformed JM109DE3 cells induced with either IPTG (4.3 mM) or Lactose (430mM). Activity of the crude extracts showed that there was less specific activity in the IPTG induced cell extract than in the lactose induced extract. The lactose induced cells continued to grow after induction but the IPTG induced cells did not grow significantly. The extracts were purified through 0.5 ml Ni-NTA Agarose columns and the specific activities of the purified b-galactosidase was similar. Also, on a per cell basis the yield was also similar between IPTG and Lactose induced purified proteins. This confirms the observation from last year that IPTG is an inhibitor of b-galactosidase activity and adds evidence that the IPTG inhibitor is removed upon purification through the Ni-NTA Agarose column.

Group 7

Lashauna Jackson and Samatha Tweed

The goal of this project was to express b-galactosidase in E.coli cells and to compare levels of expression in IPTG induced cells to the level of expression in uninduced cells.

Group 9

Eli Roberson and John Schrimsher

The goal of this project was to compare the effects of transformation on the expression of b-galactosidase. Chemically-competent and Electro-competent cells were transformed with pcDNA3.1His B/Lac Z/SD plasmid. It was found that 1350 V killed the electrocompetent cells but that 1250 V allowed 30% to survive and take up the plasmid. Transformed cells were plated on LB Amp plates and colonies were streaked on additional plates to isolate pure cultures of the transformed cells. Cells were grown in LB media and extracts taken and assayed for b-galactosidase activity at various time points. The results showed no significant difference between the b-galactosidase activity, on a per cell basis, of chemically transformed cells and electroporated cells.