Microbial Informatics

• Homework 3 is due today
• Have posted some guide lines to follow for Project 1
• No class on Tuesday (Fall break); Next Friday, the first hour of class will be a lecture
• Start programming discussion next Thursday!
• Read The Art of R Programming (Chapter 1: Getting Started)

• Type I and Type II errors
• ANOVA is a generalized form of the t-test (Parametric)
• Kruskall-Wallis is a generalized form of the Wilcox-test (Non-parametric)

• Regression
• Correlation

• You have isolated an enzyme from a novel bacterium that you are convinced will solve the world's energy problems. But first you need to characterize its kinetics on a variety of substrates.
• For a variety of substrate concentrations, you run a colormetric assay and quantify the activity (amount of product formed per unit time) for each substrate concentration. Your data look like so...

• You recall from your undergraduate biochemistry class doing a lab on Michaelis-Menton kinetics and that there is a model that looks like this:

\[\frac{dP}{dt}=V_o=\frac{V_{max}[S]}{K_m+[S]}\]

• How do you figure out \(V_{max}\) and \(K_m\)?

We can make a Lineweaver-Burk plot

\[\frac{1}{V_o} = \frac{K_m + [S]}{V_{max}[S]}\]

or...

\[\frac{1}{V_o} = \frac{K_m}{V_{max}}(\frac{1}{[S]}) + \frac{1}{V_{max}}\]

\[\frac{1}{V_o} = \frac{K_m}{V_{max}}(\frac{1}{[S]}) + \frac{1}{V_{max}}\]

• If we can fit a line through the data we can get our Vmax from the intercept and our Km from the slope
• Cue... linear regression!

\[y_i = \alpha + \beta x_i + \epsilon_{ij}\]

• We need to fit this model to estimate the values of \(\alpha\) and \(\beta\)
• We'll come back to talking about \(\epsilon\) later

• Minimize the distances between the line we fit through the data and the observed values.
• This distance is the residual - minimize the squared residuals...

\[SS_{res} = \sum(y_i - (\alpha + \beta x_i) )^2\]

• Recall how to minimze a function?

\[\hat{\beta} = \frac{\sum(x_i-\bar x)(y_i-\bar y)}{\sum(x_i-\bar x)^2}\] \[\hat{\alpha} = \bar y - \hat{\beta}\bar x\]

xbar <- mean(invS)
ybar <- mean(invV)

beta <- sum((invS-xbar)*(invV-ybar))/sum((invS-xbar)^2)
alpha <- ybar - beta * xbar

plot(invS, invV, lwd = 3, col = "blue", pch = 19, xlab = "1/[Substrate] (ml/mg)", 
    ylab = "1/Vo (min)")
abline(b = beta, a = alpha, lwd = 3)

\[\frac{1}{V_o} = \frac{K_m}{V_{max}}(\frac{1}{[S]}) + \frac{1}{V_{max}}\]

\[\hat{\alpha} = \frac{1}{V_{max}} = 1.1252\] \[\hat{\beta} = \frac{K_m}{V_{max}} = 71.2447\]

So we get a Vmax of 0.89 and a Km of 63.32

lm(invV~invS)

## 
## Call:
## lm(formula = invV ~ invS)
## 
## Coefficients:
## (Intercept)         invS  
##        1.13        71.24

\[\hat{\beta} = 0\]

• How to test?

\[t=\frac{\hat{\beta}}{SE(\hat{\beta})}\]

with n-2 degrees of freedom

summary(lm(invV~invS))

## 
## Call:
## lm(formula = invV ~ invS)
## 
## Residuals:
##     Min      1Q  Median      3Q     Max 
## -0.3986 -0.0555  0.0255  0.0765  0.2485 
## 
## Coefficients:
##             Estimate Std. Error t value Pr(>|t|)    
## (Intercept)   1.1252     0.0477    23.6  5.5e-15 ***
## invS         71.2447     1.6892    42.2  < 2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.165 on 18 degrees of freedom
## Multiple R-squared:  0.99,   Adjusted R-squared:  0.989 
## F-statistic: 1.78e+03 on 1 and 18 DF,  p-value: <2e-16

What do you notice about the fit?

• The R squared value!
• Represents the percentage of the variance in the data that is reduced by going from a one parameter model (\(\alpha\)) to the linear, two parameter model

mm <- function(S, vmax, Km){
    Vo <- vmax * S / (Km + S)
    return(Vo)
}

nonlinear.fit <- nls(dPdt ~ mm(S, v, k), start=c(v=1, k=60))

summary(nonlinear.fit)

## 
## Formula: dPdt ~ mm(S, v, k)
## 
## Parameters:
##   Estimate Std. Error t value Pr(>|t|)    
## v   0.8122     0.0242    33.6  < 2e-16 ***
## k  50.7066     4.6200    11.0  2.1e-09 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
## 
## Residual standard error: 0.0232 on 18 degrees of freedom
## 
## Number of iterations to convergence: 3 
## Achieved convergence tolerance: 5.56e-06

plot(S, dPdt, lwd = 3, col = "blue", pch = 19, xlab = "1/[Substrate] (ml/mg)", 
    ylab = "1/Vo (min)")
lines(S, mm(S, vmax = lin.vmax, Km = lin.Km), col = "red", lwd = 2)
points(S, predict(nonlinear.fit), type = "l", lwd = 2)

Wikipedia

• Parametric
• Linear relationship

\[r=\frac{(x_i-\bar x)(y_i-\bar y)}{\sqrt{\sum(x_i-\bar x)^2\sum(y_i-\bar y)^2}}\]

Sbar <- mean(S)
dPdtbar <- mean(dPdt)
r <- sum((S-Sbar)*(dPdt-dPdtbar))/sqrt(sum((S-Sbar)^2)*sum((dPdt-dPdtbar)^2))
r

## [1] 0.915

cor(S, dPdt, method="pearson")

## [1] 0.915

cor.test(S, dPdt, method="pearson")

## 
##  Pearson's product-moment correlation
## 
## data:  S and dPdt
## t = 9.62, df = 18, p-value = 1.615e-08
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  0.7939 0.9663
## sample estimates:
##   cor 
## 0.915

cor(S, dPdt, method="spearman")

## [1] 0.9789

cor.test(S, dPdt, method="spearman")

## 
##  Spearman's rank correlation rho
## 
## data:  S and dPdt
## S = 28, p-value = 6.521e-06
## alternative hypothesis: true rho is not equal to 0
## sample estimates:
##    rho 
## 0.9789