Final Exam

Physical Biology of the Cell

Student Name: ..........................................................

Student ID #: ........................................

January 15, 2013

PBC Course Final Exam 1

Physical Biology in Flow

Problem 1.1 (5 pts): Many cells in the body exist in an environment of flow. For example, the
endothelial cells in the 2D monolayer that lines the blood vessels exist in contact with flowing
blood. Do cells that exist in 3D tissues exist in flow? Specifically, do cells in (a) cartilage or (b)
bone exist in flow? Answer yes or no to both, and write 2-3 sentences, and provide a sketch to
justify your answer.

Problem 1.2 (5 pts): Most cells have a specialized organelle that functions to sense flow. Name
this organelle, and provide 2-3 sentences and a sketch to describe this organelle.
PBC Course Final Exam 2

Adhesion
Problem 2.1 (5 pts): If an adhesive interaction (cell-matrix) between an adhesion receptor and a
matrix protein can withstand force F, it is often found that a force of N F is required to pull the cell
away from the matrix, where N is a number between, say, 10 and 50. Explain with a few sentences
and a diagram how this is possible.

Rate Expressions
Problem 3.1 (15 pts): Consider a microbial bioreactor in a continuous stirred tank mode. As
derived in class, the concentration of cells Cc is given as a function of dilution D by equation:
✓ ◆
1 D ⇤ KS
Cc = C S0
YSc µmax D

Where:
YSc is a yield coefficient
CS0 is the concentration of substrate in the in-flow
CS is the concentration (variable) in the well-mixed reactor and out-flow
D is the dilution = vV0 , where v0 is the volumetric in-flow rate and V is the bioreactor volume
KS and µmax are the Monod constants

From this, derive an equation to give the maximal rate of cell production by the bioreactor, where
the rate of cell production is given by DCc . Show the derivation. (use other side of this page)
PBC Course Final Exam 3

Transcription Factor Structure

Problem 4.1 (3 pts): Which groove of the DNA do Transcription Factors most often bind to and
why?

Problem 4.2 (2 pts): Which type of protein secondary structure is most often used by transcription
factors to bind to DNA and why?

Problem 4.3 (3 pts): Name at least 3 different transcription factor families.

 The EMBO Journal Vol.16 No.15 pp.4689–4697, 1997 domain
a dimer
was det
(MIR) m

Crystal structure of PHO4 bHLH domain–DNA

acid seq
those o
dimensi

complex: flanking base recognition

Each pe
and B.
designa
PBC Course Final Exam 4 of the U
has one
1994).
symmet
ing syst
Problem 4.4 (2 pts): Identify one specific and one non-specific TF - DNA interaction in the are as d
following figure: Flanking base recognition by PHO4
et al. (1
The
helices,
that con
DNA as
Toshiyuki Shimizu, Atsuki Toumoto, (O’Neill et al., 1996). The ki parallel

Kentaro Ihara, Masato Shimizu1, PHO85 complex is down-reg 4690

Yoshimasa Kyogoku2, Nobuo Ogawa3, T.Shimizu et al.

PHO81 in the starvation of p
Yasuji Oshima3 and Toshio Hakoshima4 1994).
The PHO4 protein consists
Department of Molecular Biology, Nara Institute of Science and (Yoshida et al., 1989) and h
Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-01, (Ogawa and Oshima, 1990). T
1Biomolecular Engineering Research Institute, Furuedai, Suita,
Osaka 565, 2Institute for Protein Research, Osaka University,
is a DNA binding domain com
Yamadaoka, Suita, Osaka 565 and 3Faculty of Engineering, followed successively by a he
Osaka University, Yamadaoka, Suita, Osaka 565, Japan that was first identified in an
4Corresponding author binding factor and then in othe
e-mail: hakosima@bs.aist-nara.ac.jp et al., 1989). The bHLH mot
d the diverse regulatory proteins fou
, cyan;
The crystal structure of a DNA-binding domain of to human. In a class of reg
PHO4 complexed with DNA at 2.8 Å resolution revealed motif is followed by a Leu
that the domain folds into a basic–helix–loop–helix carboxy-terminus. These bH
(bHLH) motif with a long but compact loop that mediate dimerization that r
e other contains a short a-helical segment. This helical struc- both heterodimers and homod
’Amaré ture positions a tryptophan residue into an aromatic sequence targeted by several
bHLH cluster so as to make the loop compact. PHO4 binds to be a 5 -CANNTG-3 eleme
nd E47 to DNA as a homodimer with direct reading of both 1990) known as the E-box (B
uctures Fig.the core E-box
4. Summary sequence
of contacts of PHO4 CACGTG
residues with and
DNA itsbases
3 -flanking
and for review), in which the
viations phosphate groups. Schematic summary of the
bases. The 3 -flanking bases GG are recognized bybase and phosphate specified by each protein. PHO
contacts made by each monomer. The DNA is represented as a
esidues Arg2 and His5. The residues involved in the E-box
cylindrical projection with phosphates indicated by circles. The E-box
to its upstream activation s
he loop recognition
bases are stippled and are His5, flanking
recognized Glu9 bases and are Arg13,
hatched.as already
Base pair E-box motif in the promoter
Fig. 5. The base-specific interactions. The views are
amino- reported
recognitions are for bHLH/Zip
indicated proteins
by bold-lined arrows, MAX and USF, and
and phosphate et al., 1994; Vogel et al., 19
1 in the recognitions by thin-lined
are different fromarrows.
thoseThe weak interaction
recognized is shownproteins
by bHLH by 1992; Hayashi and Oshima,
, which dashed-lined
MyoD and arrows. All contacts
E47, although are via
PHO4side chains.
is a bHLH protein. et al., 1993) genes via the
t of the Keywords: crystal structure/E-box recognition/flanking Oshima, 1990). The recogniti
B-chain by base
PHO4 recognition/helix–loop–helix
protein (Fisher and Goding, motif/PHO4
1992). Similar be classified into two type
mmetry- contacts were also observed in the crystals of MAX, CACGTT (type 2). The type
around MyoD and E47 complexed with DNA. be more efficient than the typ
ith any It has been shown by mutation analysis (Dang et al., (Fisher et al., 1991; Fisher an
around Introduction
1992) that anOctober
Wednesday, Arg residue
10, 12 at position 13 confers specificity expression (Hayashi and Osh
ecogni- for Transcription
CACGTG (class of a set B)
of theversus
genesCAGCTG
relevant to the (class A)
phosphat- and Goding (1992) showed t
stalline E-boxes. Interestingly,
ase (PHO) system inthe thePHO4
yeast protein, like the cerevisiae
Saccharomyces bHLH/ were also involved in dete
e loop, Zipisproteins, has an Arg residue at this position,
regulated by intracellular levels of the essential nutrient despite binding. Moreover, Ogawa et
flexible its phosphate
lacking a Leu-zipper.
(Oshima, 1991;The other
Johnston bHLH andproteins
Carlson,have 1992afor the UAS sequences of sever
arboxy- hydrophobic residuegenes
review). Several (Figure have1A).been This Arg13 of
identified in PHO4
the PHO sensus sequences of PHO4 b
ifferent makes a direct, symmetrical contact
system. Those under regulation of phosphate are PHO5 with the central flanking bases, GCACGTGG
van der G(1L /1R ) ofp60,
(encoding the E-box
a major(Figure
fraction 5Cofand D), in a acid
repressible manner phos- TTT for type 2. Hence, imp
 PBC Course Final Exam 5

Measurement Techniques
Problem 5.1 (2 pts): Indicate below which methods can be used to study protein - DNA interac-
tions:
2 EMSA
2 SELEX
2 Phage Display
2 Yeast One-Hybrid (Y1H)
2 Yeast Two-Hybrid
2 PCA
2 PBMs

Problem 5.2 (3 pts): Explain the purpose of the control performed in lane 5 of the EMSA gel
EMSA - Schematic
below (the lane containing the Antibody). How is this type of control generally called?

Source: www.piercenet.com
Wednesday, October 10, 12
PBC Course Final Exam 6

Microfluidics
Problem 6.1 (5 pts): What are the main advantages of microfluidics over current fluid handling
approaches used in biology? Explain why these advantages are important.

Problem 6.2 (5 pts): Explain why mixing is often challenging on a microfluidic device. Name 2
commonly used microfluidic mixers.
PBC Course Final Exam 7

Thermodynamics
Problem 7.1 (5 pts): Write down the ODE describing the change in [AB] as a function of time for
the following system:
k k3
*1 [AB] + [C] *
[A] + [B] ) ) [ABC]
k2 k4

d[AB]
Problem 7.2 (3 pts): Solve the following ODE (show all the steps): dt
= kof f [AB]
PBC Course Final Exam 8

Problem 7.3 (10 pts): You are running an ELISA in the lab and you want to know how long you
need to incubate the Antigen with the Antibody. You assume that the observed association kinetics
follows the following equation:

[A][B]0 (kon [A]+kof f )t

[AB] = 1 e
[A] + KD

Your antigen is at a concentration of 1nM and you know the association and dissociation rates for
your particular antibody-antigen pair, which are: kon = 1x105 M 1 s 1 and kof f = 3x10 4 s 1

How long will it take to reach 90% of the equilibrium concentration of [AB]?
PBC Course Final Exam 9

Transcription Factor Specificity

You obtained the following Matrix from a SELEX experiment for a particular Transcription Factor:

PO 1 2 3 4 5 6 7 8 9
G 44 9 96 38 69 97 94 3 80
A 34 13 2 3 25 1 2 17 7
T 13 21 1 55 4 1 3 22 9
C 9 57 1 4 2 1 1 58 4

Problem 8.1 (2 pts): What is the consensus sequence for this Transcription Factor?

problem 8.2 (15 pts): Plot position 4 as a ”sequence logo” with bits as the unit for the y-axis. you
can assume a 50% GC content. Show all intermediate steps!
PBC Course Final Exam 10

Statistical Mechanics
You have the following standard reaction between a receptor (R) and ligand (L):
k on
[R] + [L] *
) [RL]
kof f

Problem 9.1 (15 pts): Setup the statistical mechanics model for this system.
Macrostates of the System:

Energy of the system:

Enumerate Microstates:

Statistical weight of each microstate:

Probability of the unbound state:

 PLoS BIOLOGY
PBC Course Final Exam 11
Plasticity of the cis-Regulatory Input
Function of a Gene
Promoter Architecture
Avraham E. Mayo¤, Yaakov Setty, Seagull Shavit, Alon Zaslaver, Uri Alon*
Problem 10.1 (5 pts): In the following figure, what logic functions do the input-output functions
Logic Gate
Departments of Molecular Cell Biology and Physics of Complex Systems, The Weizmann Institute of Science, Rehovot, Israel
M1 and M2 most closely resemble? Write down the truth tables for these two logic functions. Plasticity of a Gene CRI Equivalent:
The transcription rate of a gene is often controlled by several regulators that bind specific sites in the gene’s cis-
regulatory region. The combined effect of these regulators is described by a cis-regulatory input function. What
WT
determines the form of an input function, and how variable is it with respect to mutations? To address this, we employ
the well-characterized lac operon of Escherichia coli, which has an elaborate input function, intermediate between
Boolean AND-gate and OR-gate logic. We mapped in detail the input function of 12 variants of the lac promoter, each
with different point mutations in the regulator binding sites, by means of accurate expression measurements from AND/OR
living cells. We find that even a few mutations can significantly change the input function, resulting in functions that
resemble Pure AND gates, OR gates, or single-input switches. Other types of gates were not found. The variant input
functions can be described in a unified manner by a mathematical model. The model also lets us predict which functions
cannot be reached by point mutations. The input function that we studied thus appears to be plastic, in the sense that

M1
many of the mutations do not ruin the regulation completely but rather result in new ways to integrate the inputs.
Citation: Mayo AE, Setty Y, Shavit S, Zaslaver A, Alon U (2006) Plasticity of the cis-regulatory input function of a gene. PLoS Biol 4(4): e45.

readily a network can learn new computations in a new

Introduction
environment.
Much of the computation performed by transcription To address this, we study the plasticity of the lac input
networks occurs in the DNA cis-regulatory region (CRR) of function. We measured the effects of point mutations in the

M2
each gene. Most genes are regulated by multiple regulators lac promoter region on its input function. We find that the lac
(inputs) that bind their CRR. The way that these inputs input function is quite plastic: even a few point mutations can
combine to determine the rate of transcription is described significantly change the CRIF, leading to input functions that
by the cis-regulatory input function (CRIF) of the gene. Well- resemble pure AND gates, OR gates, and single-input
studied examples include input functions that govern switches. A mathematical model explains these results and
developmental genes [1–4] at specific locations and times, lets us predict which types of gates can and cannot be
when certain combinations of regulators are active. The obtained with point mutations.
CRIFs are often described using Boolean functions such as
AND- and OR-logic gates [4–15], although graded [8,16–18] Results
input functions are also known to occur.
Figure 3. Inputmap
Recently, a high-resolution Function of CRIF
of the the Wild-Type LibraryRegion
lac Promoter
of a well- of Variants
and Twoof the lac CRR
Variant CRRs
characterized The input
gene functions
system, the describe
lac the promoter
operon [19–21] activity
of asTo study the
a function effects
of the of point mutations
concentrations of the twoininducers
the regulator
IPTG (in lM) and cAMP (in mM), measured i
Wednesday, May 18, 2011
midexponential
Escherichia coli, was obtained, using growth in angene-expression
accurate automated fluorimeter binding
by meanssites of
ofathe lac CRR, we fusion
GFP-promoter constructed a random
on a low-copy library (A) Surface plot of log wild-type inpu
plasmid.
measurements from function.
living(B)cells
Contour plot of
[8]. The lac wild-type
CRIF hasinput of CRR
two function. mutants. The
(C) Schematic drawinglibrary was the
showing based on thethresholds
activation 113-bp and the four plateaus of the inpu
function. (D–F) Same for strain U340, with an OR-like input function.
regulatory region of(G–I)
theSame for strainfrom
lac operon U339wild-type
with an AND-like
E. coli. input function. The plateaus of th
inputs, corresponding to the two regulators of the system,
input functions are marked by roman numerals in (C, F, and
cAMP receptor protein (CRP) and LacI. The CRIF was found Each CRRI). variant
Plateau Icontained
occurs at zero concentration
between three to ofnine inducers,
pointplateau II at saturating cAMP and zer
IPTG, plateau III at saturating IPTG and zero cAMP, and plateau IV occurs when both inducers are saturating.
to be a rather DOI: intricate function, intermediate between mutations in selected locations in the regulator binding sites
10.1371/journal.pbio.0040045.g003
Boolean AND-gate and OR-gate logic [8] (see Figure 1 for (Figure 2). There were at most four mutations in the O3 site
all 16 possible two-input Boolean logic gates). Unlike pure
Boolean gates, which have two plateau levels (high and low) described by the ratios of the three plateaus (plateaus I, I
Academic Editor: Arthur D. Lander, University of California Irvine, United States of
and one threshold per input, the lac CRIF has four different America and III defined in Figure 3) to the fully induced platea
plateau levels and two thresholds per input [8]. Received January 24, 2005; Accepted (Plateau
DecemberIV): is the ratio
p1 Published
8, 2005; March of
28, expression with no inducers t
Here, we ask which changes in a CRIF can be caused by a 2006 expression with both saturating inducers, and p2 and p3 ar
few point mutations in the regulatory region and which DOI: 10.1371/journal.pbio.0040045 the ratios of expression with only one inducer (IPTG o
changes cannot. This question is related to the way in which Copyright: ! 2006 Mayo et al. This iscAMP, respectively)
an open-access to expression
article distributed under the with both. This defines a 3
the input function can be shaped by evolutionary selection terms of the Creative Commons Attribution License, which permits unrestricted
Danyphenotype space or CRIF
use, distribution, and reproduction in medium, provided the original author space (Figure 5B). Pure logi
[1]. It is believed that gene networks can ‘‘learn’’ new and source are credited. gates lie at the vertices of this space. For example, pure AND
computations on an evolutionary timescale by means of
Abbreviations: CRIF, cis-regulatory gates have CRP,
input function; coordinates
cAMP receptor ([p 1, p2, p3] ¼ [0,0,0]), pure OR gate
protein;
mutations [22–25]. Changes are mainly due to point CRR, cis-regulatory region; GFP, green fluorescent protein; IPTG, isopropyl-D-
mutations, gene duplications, and rearrangements [26–28]. thiogalactoside; RNAp, RNA polymerasehave coordinates (0,1,1), and ‘‘dysfunctional gates’’ (in whic
The degree to which mutations can change the computation, * To whom correspondence should all plateaus
be addressed. areurialon@weizmann.ac.il
E-mail: equal either to 1 or 0) have coordinates (1,1,1
without ruining the essential function, may be termed as shown in Figure 5B. Figures 5A and 5B show how uniforml
¤ Current address: Department of Biological Chemistry, University of Michigan
‘‘plasticity’’ [1,29–32]. The larger the plasticity, the more Medical School, Ann Arbor, Michigan, distributed
United States ofpoints
America in parameter space result in a nonuniform

distribution of phenotypes in CRIF space. The phenotype

PLoS Biology | www.plosbiology.org 0555 areApril
all2006 | Volume to
confined 4 | Issue 4 | e45
a restricted region of CRIF space. Thi
indicates that some CRIFs cannot be reached by poin
mutations in the CRR. The unreachable CRIFs include th
EQUAL gate, in which expression occurs when neither, o
both, inducers are present, but not if only one is present. A
additional ‘‘forbidden’’ CRIF is an XOR gate, where expres
sion occurs when only one, but not both, inducers ar
present. All ten forbidden forms are noted in Figure 1.
In contrast to forbidden CRIFs, some CRIF forms lie i
Figure 4. Input Functions of lac CRR Variants
dense regions of the design space and, thus, can be readil
The input functions are ordered from top left to bottom right to range
from those that most resemble OR gates to those that most resemble
reached by point mutations in the CRR. These function
include AND gates, OR gates, and single-input switches.
PBC Course Final Exam 12

Extra Credit Questions

Extra Credit 1 (5 pts): Why is it important to measure individual cells rather than take an average
measurement of a large population of cells?

Extra Credit 2 (5 pts): Describe in detail how the NAPPA method works (include a schematic).
PBC Course Final Exam 13

Extra Credit 3 (5 pts): Explain in detail what is meant by base interdependence (or non-independence).

Extra Credit 4 (5 pts): Explain why transcriptional regulatory networks play an important role in
evolution. Explain why mutations in cis elements are more suitable for generating small adaptive
changes than mutations in trans elements.
PBC Course Formula Sheet 1

Thermodynamics
k
on
[A] + [B] *
) [AB]
kof f

[A][B] kof f
KD = = = KA 1
[AB] kon

[A]
YB =
KD + [A]

[A]Bmax
YB =
KD + [A]

ln(2)
t1/2 =
kof f

kof f t
[AB] = [AB]0 e

[A][B]0 (kon [A]+kof f )t

[AB] = 1 e
[A] + KD

G= RT ⇤ ln(Keq )

G
Keq = e RT
PBC Course Formula Sheet 2

R is the gas constant:

1 1
R = 8.31451JK mol

1 1
RT = kb T ⇤ NA ⇠ 2.4790kJmol ⇠ 0.592kcalmol

Transcription Factor Specificity

Genome Lengths:
E. coli = 5 Mbp
S. cerevisiae = 12 Mbp
D. melanogaster = 120 Mbp
H. sapiens = 2.9 Gbp
l
Y
P (s) = P (si )
i=1

✓ ◆
1
I(wn ) = log = log(P (wn ))
P (wn )

n
X
H(x) = P (xi )logb P (xi )
i=1

X
H(i) = f (b, i)log2 f (b, i)
b

Rseq (i) = 2 H(i)

PBC Course Formula Sheet 3

height(b, i) = f (b, i)Rseq (i)

X ✓ ◆
f (b, i)
Iseq (i) = f (b, i)log2
b
P (b)

P
l
I(si )
P (s) = 2 i=1

X
Gs = Gbsi ,i
i

1
P (s) = Gs /RT
e +1

1
P (s) =
e Es µ + 1
Es = Gs /RT
✓ ◆
[T F ]
µ = ln
KD,ref

windows
Y
Pocc = 1 (1 Ps )
s=1
PBC Course Formula Sheet 4

Y ✓
windows
1
◆
Pocc = 1 1 Gs /RT
s=1
1+e

Statistical Mechanics
Sampling with Replacement:

nk

Sampling without replacement:

n(n 1)(n 2) . . . (n n + 1) = n!

Permutations
n!
n(n 1)(n 2) . . . (n k + 1) =
(n k)!
n!
P (n, k) =
(n k)!

Combinations:
✓ ◆
n n!
C(n, k) = =
k k!(n k)!