Cost of Unneeded Protein in E.coli is
Reduced after Several Generations in
Exponential Growth
Irit Shachrai,Alon Zaslaver,Uri Alon, and Erez Dekel
Murugappan Nachiappan
April 16, 2015
Roadmap
 Introduction
 Protein Cost and Impact
 Experiments
 ppGpp Systems
 Results
 Future Research
Introduction
Introduction
 E. coli serves as host for protein synthesis
 Protein cost: E. coli express unneeded proteins during
process of synthesis
 Rate of protein cost varies during exponential growth phase
 After several generations cells enter a phase with much
reduced cost
 ppGpp system and ribosomal activity are studied with
respect to protein cost
Protein Cost
and Impact
Protein cost and Impact
What is Protein Cost or Burden?
 Expression of unneeded protein in E. coli leads to
reduced cell growth rate.
 “Fractional reduction in growth rate due to limited cell
resources such as ribosomal capacity”
 This diverts the resource from producing other
beneficial proteins
Study of Protein Cost
Understanding protein cost is important for improving the
use of cells to produce desired proteins.
 Dong H, Nilsson L and Kurland (1996) experimental
measurements showed cost as a function of the levels
of the unneeded protein is not a linear function but
rather grows faster than linear.
 Stoebel et al.(2008) studied sources of protein cost in
E. coli suggested that cost originates in the production
process and not in the products.
Background

: Small DNA molecule within a cell that is physically
separated from chromosomal DNA, can replicate
independently
 Applications: researchers grow bacteria containing a
plasmid harboring the gene of interest. Just as the
bacterium produces proteins to confer its antibiotic
resistance, it can also be induced to produce large amounts
of proteins from the inserted gene.
Experiment
System for Measuring Protein
Cost
 Unneeded Protein : GFPmut3 (Green Fluorescent Protein)
 Inducer: Anhydrotetracycline – aTc C22H22N2O7 • HCl
 System with several plasmids identical except for point mutations in the
ribosomal binding sites of gfp gene.
 Bacteria were grown overnight in a defined medium, M9 + 0.2%
glycerol
 Cell density (OD) and GFP fluorescence were measured every 8 min in
a robotic system in 96-well plates.
 Induced strain was compared to a strain with the same plasmid but
without aTc in alternating wells on the 96-well plate.
 Growth rate was evaluated at each time point by the logarithmic
temporal derivative of the OD, g = d(log(OD))/dt.
Initial Observations
 Growth rate of E. coli cells
producing Green Fluorescent
Protein GFP, an unneeded
protein is measured
 High protein cost observed in
early exponential growth
 After a few generations still in
exponential growth, cells enter
a phase in which cost is
reduced.
 Though the cost is low, GFP
accumulation continues to
increase.
Further Experiments
Experiment was repeated in E. coli cells transformed with 5
variants of the plasmid that had mutated RBSs and thus
produced GFP at different rates ranging from about 5% to 15%
of total cell protein.
Variants followed the same pattern:
 Phase 1: exponential growth show a high cost of GFP
production
 Phase 2: Cost reduces to near zero
 Amount of GFP produced has a slight effect on the duration
of phase 1: Phase 1 lasts for 2 generations in the variant with
lowest expression and three generations in the variant with
highest expression.
Experiment Observations
Measured GFP production rate of the
induced and un-induced bacteria. GFP
production is high during the first growth
phase and further increases as the
bacteria enters into the second growth
phase.
 For 3 generations, cells
continued their exponential
growth and increased their GFP
production
 Beyond this, cost of GFP
production reduced to a very
low level.
Experimental Results
Circles are data from plasmids with variant ribosomal binding sites (RBSs) in the GFP gene along
with standard errors from 48 parallel measurements in 96-well plates.
Growth in Richer and Poorer Medium
 Rich Medium:
 M9C  M9 medium + Amino acids
 Same results observed only difference is it takes more
generations (6) to reach phase 2.
 Poor Medium:
 Diluted M9 medium
 Similar results as M9 observed
 Observation:
 Time to reach Phase 2 depends on concentration.
 Higher concentration leads to more growth rate.
Background

: large and complex molecular machine,
found within all living cells, that serves as the site of
biological protein synthesis.
 Application: Serves to convert the instructions found in
messenger RNA into the chains of amino-acids that make
up proteins.

is bacterial transcription initiation factor that
enables specific binding of RNA polymerase to gene
promoters.
Ribosomal Gene
 Cost of protein production
thought to be dependent on
ribosomal genes.
 Activities of 4 ribosomal RNA
promoters – rrnA, rrnB, rrnC,
rrnH were measured.
 Ribosomal promoters are
highly active to supply more
ribosomes in Phase 1
 Ribosome production
reduced to for balanced
growth in Phase 2
ppGpp Systems
 ppGpp: Guanosine pentaphosphate is an alarmone which is
involved in the stringent response in bacteria, causing the
inhibition of RNA synthesis when there is a shortage of
amino acids present.
 Alarmone: molecule synthesized due to harsh environmental
factors in bacteria.
 Stringent Response: causes cells to divert resources from
growth of cells to secretion amino acid.
 Ribosomal gene regulation: reduced cost occurs when
ribosomes are not only limiting factors for cell growth.
ppGpp system is required for growth with reduced cost.
Experiment based on ppGpp
systems
 Bacteria starts with low level of ribosome and ppGpp
 Ribosome production is maximal and limits the growth
 After 3.5 h, ribosomes increases to a level that they are not only
limiting factor
 Growth Limitation shifts to non ribosomal proteins.
 ppGpp increases as there are excess of ribosomes leading to
stalled ribosomes
 Hence ribsomal protein concentration reduces and ppGpp level
increases till then
 After 7 h, ribosomal protein count is so low, it starts limiting growth.
Hence ppGpp again reduces.
Experiments based on ppGpp
Regulation
(A) Ribosome concentration
(B) ppGpp concentration
(C) Limiting factors:
+ve: ribosomal limit
-ve: nonribosomal limit
Growth begins with ribosomal
limitations leads to balanced
growth
(D) Bacteria cell count:
increases in steady state
Experiments based on
ppGpp Regulation
 Growth limitation in balanced
growth rapidly switches
between ribosomes and nonribosomal proteins.
 Protein cost depends on
ribosomes, it is high in the
first phase when ribosomes
are limiting and lower in later
phase where ribosomes are
limiting only part of the time.
Result
Result
 Ribosomes are not the only limiting factor:
 When cells reach balanced growth non-ribosomal protein
is limiting along with ribosome.
 Unneeded proteins has a smaller effect on cost in
balanced phase as ribosomes are not only limiting.
Future
Research
Alternatives theories for
reduced cost
 Limiting factor in first phase is not due to ribosomes but
chaperones or other cellular machinery regulated by
ppGpp.
 It’s possible for proteins or mRNAs compete as limiting
factors to add up cost.
Source:
(Tagkopoulos et al., 2008; Mitchell et al., 2009).
Thank You