Download Project proposal paper - igem

Layne Woodfin
Bacterial pseudophototaxis
Introduction
The ability to respond to different external conditions is an important theme that
manifests in one way or another in all life forms. Chemotaxis and phototaxis are two key
mechanisms utilized by a wide variety of bacteria to identify potentially harmful or beneficial
environmental conditions and direct movement away or towards said areas. Some Archaea, such
as Halobacterium salinarum, utilize a phototactic response mechanism to change their
movement pattern in response to changes in light colour and intensity (3). Enterobacteria, such
as Escherichia coli, do not have photoreceptors and as such are unable to exhibit a tactic
response to light (1). Instead, E. coli uses chemotaxis to direct its movement towards attractants
and away from repellents (1-7).
E. coli have about six randomly placed flagella about their surface that are continuously
rotating. Clockwise rotation causes the flagella to bundle together and cooperatively propel the
bacterium forward, termed “running”. Counter clockwise rotation causes the previously
mentioned bundled flagella to disperse and randomly reorient the cell. Promotion of running and
suppression of the tumbling phase, mediated by the chemotaxis signalling transduction pathway,
allow the bacterium to swim away from or towards different environments. Five different
chemoreceptors of the cytoplasmic membrane, mainly localized to the poles of the cell, bind
different ligands (attractants) to initiate the cascade. For example, the tar receptor binds Laspartate and initiates a signalling pathway that allows E. coli to swim up a concentration
gradient of this amino acid (4-7,9).
Previously, Jung et al. demonstrated that by integrating photoreceptive components from
other species into E. coli’s chemotaxis system they were able to bestow upon the enterobacteria a
system of phototaxis wherein activation of photoreceptors lead to direct activation of the
chemotaxis signalling pathway without using the chemotaxis receptors. Here, I propose a
simplified method using BioBricks for incorporating the light sensor, developed by Levskaya et
al., upstream of the chemotaxis system in E. coli in order to confer upon the bacteria a secondary
phototaxis system or “pseudophototaxis”. As shown in Figure 1, activation of a light responsive
receptor will trigger the production of the enzyme; Aspartate ammonia-lyase, hereafter referred
to as aspartase. This enzyme will catalyze a reaction in the cells to create L-aspartate, a
chemoattractive molecule. L-aspartae will be secreted by the cells and act as a beacon for other
bacteria to swim towards; forming a cluster around the cells initially activated by light as shown
in Figure 2a,b and c. OmpR-P induced expression of GFP should allow for visualization of this
dense cluster of cells, while light induced expression of mCherry will be the basis of an assay for
those cells that are theoretically producing aspartase.
Device components and designs
Two key components are at the core of this project. First is the light sensor system, part
BBa_M30109, consisting of components originally developed by Levskaya et al. and requiring
an ΔEnvZ strain of E. coli to successfully function (obtainable from the registry). The light
inducible receptor is a fusion of the light responsive domain, Cph1 (maximally responsive at
660nm), and the Histidine-kinase domain, EnvZ (capable of phosphorylating the transcription
factor OmpR) (1). Additional accessory proteins required for formation of the receptor are also
present on the device as well as ribosome binding sites (RBS), terminators and a promoter
allowing for constitutive expression. On ‘activation’ by light, the His-kinase domain is inactive.
In the dark, it is actively phosphorylating OmpR. Phosphorylated OmpR (OmpR-P) can bind the
OmpC promoter and initiate transcription of downstream genes. Because incident light cannot
directly be linked to transcription, this system will require an inverter.
Ideally, the TetR inverter system would be used however, a TetR repressible promoter is
already present on the light sensor device. In order to prevent any crosstalk between devices, the
cI lambda inverter, part BBa_Q04510, will be used instead. This inverter consists of the cI
repressor protein (LVA tagged for quick degradation) followed by the cI regulated promoter.
The device itself will be placed downstream and under the control of the OmpC promoter, part
BBa_R0082, and will itself control the transcription of aspartase (part BBa_C0083) and mCherry
(part BBa_J06504, excited at 587nm and emitting at 610nm). In the presence of OmpR-P (in the
dark), the cI repressor protein will be transcribed and translated. The repressor will bind the cI
promoter, inhibiting transcription of the aspartase and mCherry as shown in Figure 3. In the
light (OmpR is not phosphorylated), the inverter will not be active and the two protein coding
regions following it will be actively transcribed and translated as shown in Figure 4.
The second core component of the pseudophototaxis system is aspartase. This enzyme,
under control of the inverter system will be cotranscribed along with mCherry. If for any reason
the translation of the dicistonic mRNA fails, the individual genes may be separately placed under
the control of the cI lambda promoter.
The GFP generating device (part BBa_E0840, excited at 501nm and emitting at 511nm),
premade with an RBS and terminator, will be placed downstream of the OmpC promoter.
All transcripts in the system will use the registry standard RBS (part BBa_B0034) and the
transcriptional terminator; part BBa_B0015 as it is recognized as the most commonly used
terminator in the registry.
The parts and devices of this system will be put onto two different plasmids as depicted
in Figure 2d. The light sensor device and GFP generator will be placed on the medium copy
plasmid: pSB3C5. The inverter, aspartase generator and mCherry generator will be placed into
the medium copy plasmid: pSB6A1.
Experimental considerations
Aspartase catalyzes the reversible ammination of fumarate to L-aspartate in E. coli (2).
Many important question arise with respect to this enzyme and, due to the lack of experience or
referenced information on its parts list page, I have chosen to investigate these questions further.
Aspartase is already present in wildtype E. coli and given that its substrates (ammonium
and fumarate) will be present in the medium in which it’s grow, there is potential for endogenous
aspartase to use up large amounts of fumarate (2,8-10). This could potentially prevent the
additional aspartase (produced by a subpopulation of cells on exposure to light) from being able
to form a useful concentration gradient of L-aspartate. It may, therefore, be necessary to
knockout endogenous aspartase in the E. coli strain used for this experiment. In previous
research, Marcus and Halpern demonstrated that aspartase, while an important enzyme in E.
coli’s metabolism, was not essential for survival. The researchers used strains of E. coli with a
mutation allowing them to constitutively take up glutamate (due to a defect in an operator of the
glutamate permease gene). These mutant strains (Glut+) of bacteria had the ability to survive on
glutamate as a sole carbon and energy source. From these strains, they screened for mutants that
lost the ability to live solely off of glutamate. One of these was the aspartase deficient (AspA-)
C58 strain which, incapable of growing solely on glutamate, could still grow on glucose or
succinate minimal media (8).
Another key factor to the function of the proposed system is whether the L-aspartate that
will be produced and used as a chemoattractant signal will even be excreted by the cells.
Budrene and Berg previously demonstrated that E. coli inoculated onto plates containing a
minimal medium augmented with between .5 - 7 nm succinate could utilize the succinate and
excrete L-aspartate. Coincidentally, they utilized the same C58 AspA- Glut+ strain used by
Marcus and Halpern proving that aspartase was responsible for this production of L-aspartate
and, most importantly, that the mutant strain was viable on their succinate minimal medium
(9,10). Additionally, using succinate minimal media should bypass high levels of catabolite
repression that aspartase is normally subject to. In the presence of glucose, aspartase production
is highly limited due to the bacterium’s preferential use of glucose as a carbon and energy source
(2,9,10).
Based on the previously mentioned findings, it should be possible to construct AspAcells from a highly motile strain of E. coli with a system that produces aspartase on exposure to
light. When plated on a minimal succinate medium (9,10), cells exposed to light should produce
aspartase and begin making L-aspartate. On excretion of L-aspartate, a gradient of the amino
acid will form via diffusion and cause surrounding cells to migrate towards those that were
initially activated.
Device assembly
All of the parts and devices used in my design conform to BioBrick assembly standard
RFC10 and will therefore be put together using this simplified system. Under BioBrick design
standards, all parts obtained from the registry in plasmids backbones with the form:
plasmid -- 5’ -- EcoRI NotI XbaI -- GENE OF INTEREST -- SpeI NotI PstI -- 3’ -- plasmid
Cutting at the EcoRI and SpeI sites yields a product called the Front Insert;
5’ -- EcoRI NotI XbaI -- GENE OF INTEREST X -- SpeI -- 3’
Cutting at the EcoRI and XbaI sites yield a product called front vector;
plasmid --5’ -- EcoRI
XbaI -- GENE OF INTEREST Y -- SpeI NotI PstI -- 3’ -- plasmid
The front insert can be ligated onto the now open sticky ends of the front vector to create
a new plasmid with both parts. The two EcoRI sticky ends are ligated together to create a new
EcoRI site and the SpeI and XbaI sticky ends are linked to create a mixed SpeI/XbaI site;
plasmid -- 5’ -- EcoRI NotI XbaI -- GENE OF INTEREST X – SpeI/XbaI GENE OF --- INTEREST Y -- SpeI NotI PstI -- 3’ -- plasmid
The SpeI/XbaI mixed site is no longer capable of being cut. This procedure can be
repeated by cutting the new composite part as a front insert and inserting it into a different front
vector to create a plasmid with three parts and so on (11).
A motile ΔEnvZ strain of E. coli will be transformed using markerless insertion to create
an AspA- GltC- mutant strain. Strains with the desired characteristics will be easily identifiable
by plating on glutamate minimal media (where successfully mutated cells should be able to
survive while wildtype E. coli cannot) and succinate minimal media (where a group of cells,
inoculated on to a single point of the plate, should display no chemotactic response relative to
wildtype) (9,10).
Example assembly methods
The parts for the light sensor device/GFP plasmid will be transformed into E. coli in the
high copy, AMP resistant plasmid; pSB1A3. Each of the following parts will be ligated
together:
BBa_R0082
OmpC promoter
BBa_E0840
GFP generating device (includes RBS and terminator)
BBa_M30109
Light sensor generating device
1. Purify R0082, E0840 and M30109 from previously transformed E. coli using recycled
columns.
2. Set up restriction reactions to cut R0082 at EcoRI and SpeI restriction sites to create a front
insert. Cut E0840 with EcoRI and XbaI to create a front vector.
3. Run on gel electrophoresis to remove unwanted fragments.
4. Set up a ligation reaction and ligate R0082 to E0840 to create R0082-E0840.
5. Transform E. coli with ligated plasmid.
6. Purify R0082-E0840 plasmid from E. coli.
7. Set up restriction reactions to cut R0082-E0840 at EcoRI and SpeI restriction sites to create a
front insert. Cut M30109 with EcoRI and XbaI to create a front vector.
8. Run on gel electrophoresis again.
9. Set up a ligation reaction and ligate R0082-E0840 to M30109 to create R0082-E0840M30109.
10. Transform E. coli with ligated plasmid.
At this point, we can assay for light sensor function by virtue of competent cells
expressing GFP in the dark (unstimulated by 660nm light). Additionally, the device could be
sequenced or measured via PCR to confirm its structure.
11. Purify R0082-E0840-M30109 plasmid from E. coli. and move the cassette into the medium
copy plasmid: pSB3C5.
Similar steps will be taken to construct the inverter-aspartase-mCherry cassette and insert
it into pSB6A1. Transforming E. coli with both plasmids will allow for testing of the second
cassette’s functionality and the function of the system overall. Aspartase production can
potentially be tested by measuring its activity (L-aspartate concentration and ultimately a tactic
response) and assaying for mCherry production (theoretically cotranscribed). The tactic
response of E. coli can be visualized on dishes they are plated on via microscopy as described in
6,7,9 and 10.
Conclusion
Here, I have proposed a streamlined setup for bestowing upon E. coli a system of
phototaxis. Theoretically, the system will allow bacteria activated by an input of light to produce
a chemoattractant that will cause surrounding cells to migrate towards them. In this way, the
cells will essentially be swimming towards the light. This project has various useful measurable
aspects including clearer definitions of the strengths of individual promoters and ribosome
binding sites and a better characterization of the aspartase enzyme which will likely lead to
greater use of it in future projects. The registry is currently lacking in parts involved in bacterial
chemotaxis and by using aspartase to induce useful behaviours in E. coli, I suspect this trend will
be reversed. Additional enzymes that produce different chemoattractants could be useful in
future projects to affect more complex patterns of movement and a greater level of control of
bacterial localization. The inclusion of repellent molecules could further add to our ability to
control movement and create interesting dynamic systems of cells that chase or run away from
each other.
Early success in this project may merit the addition of a ribokey/ribolock system into the
proposed gene circuit. By having the production of aspartase under the control of a ribokey, it
may be possible to fine tune the levels of aspartase made and the time span in which its
translation is permitted.
A successful outcome in this project has great implications for the future, both in terms of
future iGEM projects and potentially for the broader scientific community. Future projects could
further develop the pseudophototaxis system or couple it to other systems to achieve more
complex behaviours. Ultimately, finer control over the movement of bacterial systems could be
of great use to many industries. Specifically, firms utilizing biofilms or bioreactors could find
the ability to direct bacteria by light advantageous. When threatened by something in their
environment, E. coli have been shown to form clusters giving them some increased tolerance to
toxins by virtue of lower local concentrations (7). Exploitation of this property could be very
useful and easily potentially accomplished by directing E. coli to aggregate via a pulse of light.
References
1. Levskaya, A., Chevalier, A. A., Tabor, J. J.,Simpson, Z. B., Lavery, L. A., Levy, M.,
Davidson, E. A., Scouras, A. Ellington, A.D., Marcotte, E. M., and Voigt, C. A. 2005.
Engineering E. coli to see light. Nature. 438: 441-442.
2. Guest, J. R., Roberts, R. E., and Wilde, R. J. 1983. Cloning of the Aspartase Gene (aspA) of
Escherichia coli. Journal of General Microbiology. 130: 1271-1278.
3. Jung, K., Spudich, E. N., Trivedi, V. D., and Spudich, J. L. 2001. An Archaeal PhotosignalTransducing Module Mediates Phototaxis in Escherichia coli. Journal of Bacteriology.
183: 6365-6371.
4. Webre, D. J., Wolanin, P. M., and Stock, J. B. 2003. Bacterial Chemotaxis. Current Biology.
13: R47-R49.
5. R. Tyson, S. R. Lubkin, and J. D. Murray. 1999. A Minimal Mechanism for Bacterial Pattern
Formation. Proceedings: Biological Sciences. 266: 299-304.
6. Maki, N., Gestwicki, J. E., Lake, E. M., Kiessling, L. L., and Adler, J. 2000. Motility and
Chemotaxis of Filamentous Cells of Escherichia coli. Journal of Bacteriology. 182:
4337-4342.
7. Mittal, N., Budrene, E. O. Brenner, M. P., and van Oudenaarden, A. 2003. Motility of
Escherichia coli cells in clusters formed by chemotactic aggregation. Proceedings of the
National Academy of Sciences of the United States of America. 100: 13259–13263.
8. Marcus, M. and Halpern, Y. S. 1968. The metabolic pathway of glutamate in Escherichia coli
K-12. Biochimica et Biophysica Acta. 177: 314-320.
9. Budrene, E. O., and Berg, H. C. 1991. Complex Pattenrs Formed by Motile cells of E. coli.
Nature. 349: 630-633.
10. Budrene, E. O., and Berg, H. C. 1995. Dynamics of Formation of Symmetrical Patterns by
Chemotactic Bacteria. Nature. 376: 49-53.
11. Knight, T. 2006. Idempotent vector design for standard assembly of BioBricks. MIT
Synthetic Biology Working Group.