Time Course Effect on Activity

1
Time Course Effect on Activity

• Jonathan Sims
• Jyoti Sahi
• RGT 450 Project II

2
Introduction
The aim of this project was to determine what
effect time played on the cellular activity of
the pcDNA 3.1/HisB/LacZ plasmid. A
quantitative approach was taken in order to see
how IPTG and Lactose affect a cell in regards to
its protein concentration and enzyme activity
over the course of three hours post induction.
The experiment was designed in a time-sensitive
fashion that would yield the truest form of the
data desired.
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Materials Methods

• pcDNA 3.1/HisB/LacZ plasmid was introduced into
  BL-21 (DE3) pLyse competent cells for
  transformation.
• A five mL liquid culture was grown in LB (Lurie
  Broth) with the addition of 34 mg/mL
  chloroamphenical and 50 mg/mL Amp.
• A 1/50 dilution was prepared of this liquid
  culture to a final volume of 30 mL.
• Diluted culture was grown to OD600 of 0.4 in a
  37C shaker.

4
Materials Methods

• The cultures were induced with 0.5 mM IPTG and
  50.0 mM Lactose.
• OD600 measurements were recorded every thirty
  minutes for three hours after induction.
• ß-galactosidase expression was monitored for the
  induced samples in order to determine the enzyme
  activity.
• NanoOrange Protein Assay Kit was used to quantify
  protein in the cell. The protein assays were
  viewed using the Storm 860 analyzer, and density
  of the amount of protein in each well was
  determined using computer equipment supplied in
  the lab.

5
Results Conclusions

• OD600 measurements displayed consistent growth
  for both IPTG
• and Lactose induced cells. It can be noted that
  IPTG induced cells showed more growth because the
  OD values were higher.

6
Results Conclusions

• These graphs represent the standards used to
  determine enzyme activity and protein
  concentration. Values for these two factors were
  derived using the equation noted on the graphs.

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Results Conclusions
This graph correlates cell growth, enzyme
activity, and protein concentration with respect
to time.
8

Results Conclusions
This graph correlates cell growth, enzyme
activity, and protein concentration with respect
to time.
9
Discussion

• It should be noted that both the Lactose and
  IPTG-induced cultures demonstrated increasing
  amounts of protein and enzyme activity as the
  amount of cells in the culture increased.
• The enzyme activity of the Lactose-induced cells
  increased in a linear fashion, but not quite to
  the same yield as those induced by IPTG.
• It was concluded that inducing with IPTG yielded
  the maximum enzyme activity over the course of 3
  hours.

10
Discussion

• The protein concentration over the course of
  three hours showed a linear increase when induced
  with Lactose.
• The IPTG-induced culture demonstrated the same
  increase in enzyme activity compared to the
  increase in the amount of cells. The amount of
  protein in the system seems to reach a maximum
  point before dropping off.
• The linear increase in protein concentration is
  greater for Lactose than IPTG.

References NanoOrange Protein Quantitation Kit
(N-6666), Molecular Probes, Inc. 2001