Galactosidase Expression in pcDNA 3'1HisBLacZLyseE in E' coli

1
ß-Galactosidase Expression in pcDNA
3.1/HisB/LacZ/LyseE in E. coli

• Using Variable Concentrations of Lactose and
  IPTG as Inducers
• By Michael Dodson and Sondra Massey
• Biology 450
• Dr. Rinehart
• Spring 2004

2
Procedures Used

• Transformation of plasmid into competent E. coli
  cells using AMP for selection
• Inoculation of transformed cells into LB/AMP and
  CAM for overnight growth
• Dilution and growth to 0.400 at A600
• Induction of LacZ gene using 0.3M, 0.2M, 0.1M
  concentrations of Lactose and IPTG
• Ni-NTA agarose purification
• Protein Assay
• Anaylsis

3
Introduction

• The purpose of this experiment was to see if the
  concentrations of known inducers would produce
  significantly different results of protein
  production
• To see if one was preferred over the other at
  different concentrations

4
Materials and Methods

• Cell transformation (1)
• Inoculation of cells was in LB/AMP(50mg/ml) and
  CAM(34mg/ml)
• Dilution factor of 1/50 was used in growth of
  cells to 0.400 at A600 in LB/AMP/CAM
• 0.3M, 0.2M, and 0.1M concentrations of Lactose
  and IPTG were added to individual flasks of
  LB/AMP/CAM and allowed to induce for 2 hours.
  Control sample was used also.
• Spectroscopy was done
• Cells centrifuged (5000 rpm, 20 minutes, 4C ).
• Supernatants was isolated and pellets were
  resuspended in lysis buffers (50mM TRIS-Cl, pH
  8.0, 1mM EDTA, 100mM NaCl) .
• 6 Drops of chloroform and 3 drops of 0.1 SDS
  added to each.
• Used freeze-thaw method to sheer DNA and RNA
• Part of cleared supernatant was used for total
  protein quantification
• Used Protocol 12 from The QIAexpressionist A
  Handbook for High Level Expression and
  Purification of 6X His-tagged proteins, 5th
  Edition, June 2003 pp119-121. Modifications to
  the protocol included an engineered filtration
  system using 5 ml syringe, filter paper and
  falcon tube to catch liquids.

5
Methods and Materials (cont.)

• Another modification was an extra wash with wash
  buffer.
• Serial dilutions were made with standardized
  protein BSA, beginning concentration 20µg/ml and
  ending concentration of 0.01956µg/ml and two fold
  dilutions with samples, total proteins and
  purified proteins, 1/10 and 1/100 for total
  proteins 10µl start before serial diltuions and
  1/10 start for purified proteins.
• Fluorescence mixture made (2) and added to each
  sample.
• Samples put in thermocycler for 5 minutes at 90C
  and allowed to cool at room temperature.
• Plates read in Storm860 spectrophotometer.
• SDS-PAGE(12, H2O 1.1ml, 30 acrylamide mix
  2.0ml, 1.5M Tris, pH 8.8, 1.3ml, 10 SDS 0.05ml,
  10 ammonium persulfate 0.05ml, TEMED 0.002ml)
  gel was run on purified sample at 200V/cm of gel
  after sample was in thermocycler for 4 minutes at
  96C. This was ran for 30 minutes.
• Stained for 5 minutes with Coomassie Blue,
  destained for approximately 5 minutes.

6
Spectroscopy Results After Induction
7
Results and Discussion

• After chloroform and SDS addition, samples were
  spun at 5000 rpm, 10 minutes, 4C. Not all
  pelletted. Those were transferred to microfuge
  tubes and microfuged at 13,200 rpm for 20 minutes
  total.
• Freeze-thaw method used a 40C water bath and
  -80C freezer.
• Samples were microfuged again at 13,200 rpm, 4C
  for total of 40 minutes.
• 20µl of each solution was used for total protein
  quantification.

8
BSA Standard Curve and Concentrations of Proteins
from Induction
9
Protein Amount Produced From Induction
10

• Conclusions
• We concluded that the concentrations of 0.3 M and
  0.2 M lactose give about the highest yield of
  protein expression of the three concentrations of
  lactose used.
• 0.1M IPTG gives a very similar but slightly
  higher amount of protein expression to the
  lactose while the other two concentrations of
  IPTG give a much lower amount.

11
References

• 1. Doyle, K. (1996) Promega Protocol and
  Applications Guide, 3rd Edition Promega
  Corporation. p . 46
• 2. Scopes, R.K., (1987) NanoOrange Protein
  Quantification Kit (N-6666), Protein
  Purification, Principles and Practice, 2nd
  Edition, Springer-Verlag