Agitation and Iptg

Agitation and IPTG Induction Effects on the Growth Rate and Pfu CelB Activity in Recombinant E. coli

Paul Alava and Haley sdj

Golden LEAF Biomanufacturing Training and Education Center

North Carolina State University. Raleigh, NC.

Conducted June 30. 2015

Abstract

The growth of E.coli and CelB activity was monitored in shaker flasks under different combinations of agitation and induction with IPTG. Agitation was the decisive factor in E. coli growth as absorption skyrocketed in both flasks regardless of IPTG induction although the non-induced flask did show more growth. The flasks that were not induced showed almost no CelB activity. The flask that was agitated and induced with IPTG showed very high levels of CelB activity compared to all the other flasks including one that was induced but not agitated.

Introduction

Recombinant DNA expression is a fast and effective way to produce products that an organism normally would not make. The T7 expression system used in this lab requires fours things: an expression vector, a cloned gene, a host cell, and an inducer.

The expression vector used was pET25b(+) because it has a resistance to ampicillin which allows us to know which cells have been successfully transformed, a multiple cloning site to insert the cloned gene, and a strong promoter that will allow a large number of transcripts and therefore more protein production.

The cloned gene was lacI which binds to lacO, repressing the T7 RNA polymerase transcription. This problem is fixed by our inducer IPTG (Isopropyl β-D-1-thiogalactopyranoside) which binds to lacI preventing it from binding to lacO, allowing transcription to continue. IPTG also cannot be metabolized by E.coli, meaning its concentration remains the same and so does the rate of expression of lac promoter controlled genes.

The host cell used is Escherichia coli BL21(DE3) due to its high expression of recombinant proteins. DE3 is a prophage that carries the T7 RNA polymerase.

In this lab...