Assessment of \(\beta\)-gal Activity

Introduction

The Biobuilder iTune Device lab guides you through measurement of the activity of the lacZ gene produce known as \(\beta\)-galactosidase or \(\beta\)-gal for short. The activity level of \(\beta\)-gal represents the combined effectiveness of promoter and ribosome binding site (RBS) pairs. Weak combinations produce relatively low levels of \(\beta\)-gal while strong pairs produce relatively high levels of \(\beta\)-gal. In this way, the \(\beta\)-gal measurement is a measure of performance.

Performance is measured as the level of yellow color which is produced when \(\beta\)-gal cleaves a substrate called ortho-Nitrophenyl-β-galactoside (ONPG). \(\beta\)-gal cleaves ONPG into two components: o-nitrophenol and galactose. While the galactose is colorless, the o-nitrophenol is yellow, which provides a visible signal for \(\beta\)-gal activity. A more-yellow color indicates a more-active \(\beta\)-gal system.

Getting Started

The iTune Device activity uses ten strains for testing. For each strain, you will grow a liquid culture using LB growth media, ampicillin (for selection of the plasmid carrying the correct promoter:RBS:lacZ construct), and isopropyl β- d-1-thiogalactopyranoside (IPTG) to relieve inhibition of the lacZ gene.
Strain # Registry # (promoter part) Registry # (RBS part) Relative Strength (promoter/RBS)
2-R BBa_J23115 BBa_B0035 Reference/reference
2-1 BBa_J23113 BBa_B0031 Weak/weak
2-2 BBa_J23113 BBa_B0032 Weak/medium
2-3 BBa_J23113 BBa_B0034 Weak/strong
2-4 BBa_J23106 BBa_B0031 Medium/weak
2-5 BBa_J23106 BBa_B0032 Medium/medium
2-6 BBa_J23106 BBa_B0034 Medium/strong
2-7 BBa_J23119 BBa_B0031 Strong/weak
2-8 BBa_J23119 BBa_B0032 Strong/medium
2-9 BBa_J23119 BBa_B0034 Strong/strong
Note:
All strains are constructed in the ‘TOP10’ E.coli strain of genotype: F…

Measurement

The activity of \(\beta\)-gal will be quantified using Miller Units which are calculated as follows.

Activity (Miller Units) = \(1000 \cdot \frac{Abs420}{T \cdot V \cdot OD600}\)

where A represents \(\beta\)-gal activity, Abs420 represents the measured sample asborption at 420nm in a spectrophotometer, T represents the reaction time (minutes), V represents the volume of cells in each reaction (ml), and OD600 represents the sample optical density at 600nm in a spectrophotometer.

Procedure

  • Use a spectrophotometer to measure the absorbance of your sample in 600nm light (OD600). This is a unitless number representing the turbidity of your sample which in turn indicates the density of cells present in the sample. Higher optical density indicates the presence of a higher cell count. Record the value times 10. For example, if OD600 equals 15, record 150.

  • steps…

  • Use a spectrophotometer to measure the absorption of your sample in 420nm light (Abs420). This also is a unitless number.

  • Calculate the Miller Units according to the above formula.

Miller Units Calculation

The Miller Units can be calculated using the formula. Replace the values with your own to calculate your specific \(\beta\)-gal activity level.

abs420<-0.73 #usually 0.05 to 1; ideally 0.6 to 0.9
abs600<-0.52 #usually 0.28 to 0.70
v<-0.02      #volume in ml
t<-150       #time in minutes

#miller units
mu<-1000*(abs420/(t*v*abs600))
## Activity =  467.9487 Miller Units

The increase in Miller Units as Abs420 increases can be seen in this simulation. Note the linear relationship.

abs420<-seq(0.5,1.0,by=0.01)
t<-150
v<-0.02
abs600<-0.52

#activity calculation function
mu <- function(abs420,t,v,abs600) {
  output <- 1000*(abs420/(t*v*abs600))
  return(output)
}

#run the function for values of the sequence
activity<-mu(abs420,t,v,abs600)

#create a data frame
df<-data.frame(abs420,activity)

ggplot(df, aes(x=abs420,y=activity)) +
  geom_point(shape = 21, 
             colour = "#007099", 
             fill = "#00BBFF", 
             size = 2, stroke = 1)+
  scale_y_continuous(limits=c(300,700), 
                     breaks=seq(300,700,by=50))+
  labs(x="Absorption at 420nm", 
       y="Activity (Miller units)",
       title="B-galactosidase Activity", 
       subtitle="iTune Device Lab", 
       caption="For: time (minutes) = 150; volume (ml) = 0.02, Abs600 = 0.52")+
  theme_minimal()+ 
  theme(legend.position = "none")