competition experiment

7/27/2019 Competition Experiment

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Competition between Escherichia coli and

Saccharomyces cerevisiae under resource limiting

conditions

Allen Hoffmann

Abstract: The creation of any bioreactor system is subject to the risk of contamination by

undesirable strains. This experiment simulates a contamination scenario by culturing

S.cerevisiaewith E.coliin liquid cultures and agar plates over a two week period. When

brought into direct contact, E.coligrew faster than on open media. Unusual expression of

green fluorescent protein by E.coliwas witnessed during this study. These findings indicate

a need to protect yeast cultures from E.colicontamination.


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Allen Hoffmann 8/15/13

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Introduction:

The utilization ofEscheria coliand Saccharomyces cerevisiaestrains in biotechnological

applications is well documented. The creation of bio-reactor systems allows for the large scale

cultivation of microbial populations. Genetic engineering has allowed these microbial

populations to produce compounds of industrial or medical use. For all reactor systems

contamination by other strains is seen as a primary obstacle requiring strict aseptic protocols.

This paper describes research into competition betweenE. coliand S.cerevisiaecultured together

in resource limiting conditions as a means of simulatingE.colicontamination of a yeast reactor

system.

There is a need to utilize easily identifiable strains for this experiment due to the lack of

advanced tools like florescence microscopy. The creation of plasmids expression florescent

proteins allows for visual identification of the strains being used. TheE.colistrain HB101 K-12

is offered by Bio-Rad for use with the pGLO plasmid in transformation kits. The pGLO plasmid

from Bio-Rad, widely used in college biology labs, allows for the selective expression of green

fluorescent protein (GFP) when the cells are exposed to the sugar L-arabinose in the media. This

combination allows for an easy transformation and quick visual identification.

The growth ofE.colion luria-bertani (LB) media produces significant predictable results.

The LB media however contains no fermentable sugars indicating a need to supplement the

media with dextrose in order to facilitate yeast growth (Guennadi Sezonov, Danile Joseleau-

Petit and Richard D'Ari, 2007). In order to generate a fair competition the media both strains are

being cultured on must allow for growth of both species.


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A version of the mcherry plasmid coding for red fluorescent protein (RFP) was modified

to favor AT rich codons creating the yeast-enhanced mRFP plasmid. Yeast cells carrying this

plasmid produce a purple phenotype due to overexpression of the gene. This plasmid allows for

visual identification of fungal colonies and quick determination of plasmid carrying members via

fluorescence microscopy (Sabine Keppler-Ross, Christine Noffz and Neta Dean 2008). This

strains plasmid expression creates a striking visual difference from theE.colito be used in the

experiment.

Contamination of a high density culture of S.cerevisiaebyE.coliHB101 (Biorad)

inhibited the growth rate of a high density system. Strains able to form flocculent clumps were

more tolerant of contamination. Another implication of this study is that cells facing competition

may drop plasmids that slow down cellular replication (Luclia Domingues, Nelson Lima, Jose

A. Teixeira 2009). It can be inferred from this study that a sudden drop in plasmid expression

would be an indication of those yeast cells undergoing competition for resources.

Saccharomyces cerevisiae is one of the few yeast strains capable of anoxic fermentation

of sugars (Cornelius Verduyn, et al. 1989). This may indicate the possibility of resource

partitioning within deep liquid cultures where we would expect to see yeast surviving at the

bottom of the container in a region whereE.coligrowth is unsupported. When combined with the

issue of yeast flocculation it may be possible for liquid reactors to significantly limit the growth

ofE.coli. An issue to look at in this experiment is how yeast handles contamination in aerobic

environments.


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Methods:

TheE.colistrain HB101 K12 (Biorad) was on hand for the biology lab and was

previously transformed with the PGLO plasmid.

The S.cerevisiaestrain was acquired from Dr. Neta Dean, Stonybook, NY, it was

provided previously transformed with the YemRFP plasmid as described in literature review.

Primary cultures were maintained by Dr. Mary Campbell, curator of the biological department

for Deanza Community College. Subcultures were maintained in liquid media and on agar plates

at 37C. The experiment was overseen by Dr. Brian McCauley in the molecular biology

laboratory at Deanza Community College.

Initial growth tests indicated a need to create specialized media for this experiment.

Standard LB broth was supplemented with dextrose while the sabouraud dextrose media was

supplemented with bacto-tryptone. The media was created according to manufacturers

directions and autoclaved before the sterile-filtered arabinose was added. The plates were hand

poured and incubated at 37C for 48 hours before use to insure they were free of contamination.

For each agar plate, 100 l of inoculated liquid media for each strain was transferred to

the agar plate via pipette then spread with an inoculation loop to cover half of the plate without

allowing the liquid to touch the walls of the plate or cross over the center. The plates were stored

in the incubator at 37C. Periodically the plates were removed from the incubator, placed into a

biological safety cabinet with an ultraviolet light source, and photographed using a canon digital

camera. Photographic sets were assembled for each of the plates described in the results section.


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For liquid cultures, 100l of inoculated liquid media from each liquid subculture were

added to 3.8ml of LB media or SAB media respectively and cultured in a shaking table incubator

at 37C. These hybrid cultures were then subjected to serial dilution then plated onto prepared

agar plates for observation.

Results:

Plate Media Strain Growth

(E./S.)

Fluorescence

(color)

Notes

1 LBmix1 E.coli + - GFP did not express as expected

2 LBmix1 S.cerevisiae + + red Yeast grows well on LB +dextrose

3 LBmix1 Both +E +S +red *grn Contact induced GFP expression

4 LBmix1 Both +E +S +red *grn Plate contaminated (mold) discard

5 SABmix1 E.coli Min - Expected GFP expression, min growth6 SABmix1 S.cerevisiae + + red Yeast grows well

7 SABmix1 Both **/+ +red *grn E.coli rapid takeover, induced GFP

expression on contact, robust growth

8 SABmix1 Both **/+ +red *grn E.coli rapid growth where yeast

accidentally crossed over during

inoculation, rapid GFP expression

9 LBmixrnd2 Serial Dilution

of hybrid culture

+++/+ +red initial,

rapid green

flush

Rapid expression of GFP

10 LBmixrnd2 Serial Dilution

of hybrid culture

++/+ +red *grn GFP expression 1stseen between 3

yeast colonies, photo w/ dissection

scope

11 SABmixrnd2 Both +/+ +red *grn Weak initial E.coli growth,accelerated on contact with yeast,

induced GFP on contact

12-16 The remaining plates were used to maintain subcultures throughout the experiment

Table 1: Agar plate trial results

The control plates forE.colifailed to demonstrate any expression of the pGLO plasmid.

No green florescence was observed over the life of the control plates. This indicates an

insufficient concentration of L-arabinose in the prepared mixture that was added to the agar

media.

The control plates for S.cerevisiaedisplayed a purple to pink phenotype and bright pink

fluorescence under UV illumination which corresponded to its documented behavior.


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In the aerobic environment of the incubator,E.colidemonstrated an ability to take over

the space occupied by the S.cerevisiaelawn of cells but the speed of takeover was specific to the

scenario created on the plate therefore two of the plates have been selected as visual references.

Plate 3 (LB mix1)was inoculated according to methods with both strains and allowed to

culture in the incubator; this was considered a successful inoculation due to the distinct visible

gap between the two lawns. Initially the S.cerevisiaelawn developed faster than theE.colilawn

looking visibly thicker. Four days into the trial a white edge along the S.cerevisiaelawn could be

seen indicating presence of uracil and a dropping of the plasmid from the cells. Six days into the

trial a thin green line of florescence appeared along the boundary between the two strains

indicating activation of the pGLO plasmid. Following the 6thday robustE.coligrowth is seen

along the boundary with florescence spreading backwards through theE.colilawn. While

displaying aggressive growth, theE.colidid not significantly penetrate the yeast lawn.

Plate 7 (sab mix1)was inoculated according to methods with both strains and allowed to

incubate; this inoculation was seen as unsuccessful due to crossing over of the two strains as the

liquid settled into the agar gel. The progression ofE.coligrowth was significantly faster than on

plate 3 (LB mix1); this matched with other plate examples where the two strains were brought

into direct contact early. TheE.colicells rapidly expressed green florescence and spread across

the yeast lawn over a few days.

A liquid culture of both strains was incubated for several days then subjected to serial

dilution and plated (plate 10). An exact colony count was deemed inaccurate due to the lawn of

E.colithat had set up around the yeast. The yeast colonies were few in number but clearly visible

as a purple dot surrounded by white cells. The expression of green florescent protein by the


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E.coliwas first witnessed on this plate in a colony growing between 3 colonies of S.cerevisiae

and pictured in image 1. From that point on theE.coligrew rapidly while expressing GFP in all

colonies adjacent to yeast colonies.

Image 1: spontaneous expression of green florescent protein by E.coligrowing in proximity

to three S.cerevisiaecolonies. Photograph taken with an Olympus digital camera

mounted to a dissection scope.

Discussion:

Initial growth tests indicated a need to supplement the SAB medium with tryptone, which

is found in the LB medium that supports the growth ofE.coli. On plate 3 (LBmix1) the yeast

were able to set up a lawn that did not get significantly penetrated by theE.colithroughout the

life of the plate despite media selection that should have favored E.coligrowth. TheE.coli

growing into the edge of the yeast lawn expressed GFP while the part of the lawn away from the

yeast failed to express it throughout the trial.

On plate 7 (SABmix1) theE.coliand S.cerevisiaecame into direct contact with each

other. TheE.colirapidly spread across the top of the yeast lawn and rapidly expressed GFP. It is

interesting to note theE.coligrew faster across the yeast lawn than open media and expression of


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GFP occurred primarily in sections of theE.colipopulation in direct contact with yeast cells. The

yeast darkened over time turning from purple to brown.

In liquid cultures both strains were able to survive and produce colonies when transferred

to an agar plate. A possible explanation for this observation would be anoxic conditions at the

bottom of the test tube where both strains were being cultured. If an anoxic region were present it

would allow the S.cerevisiaeto conduct anoxic fermentation on sugars found at that depth

creating a resource partition within the media.

The expression of green florescent protein inE.colidid not occur as expected. Plates

were produced using a prepared stock of L-arabinose yet the control plates did not induce

florescence. When theE.colicame in direct contact with the S.cerevisiaecolonies, a rapid flush

of green could be seen spreading through theE.colilawn. This could not be repeated on a plate

that contained no L-arabinose. The mechanism for this expression is unknown; it is possible the

yeast were taking up the sugar and passing it to theE.coliwhen brought into contact. Another

possibility would be a fermentation byproduct triggering GFP expression when it made its way

into the bacteria. Further studies into the flexibility of the pGLO plasmid or the minimum

concentration of L-arabinose required to induce GFP in this scenario would shed light on this

issue.


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Appendix A: Photographic series and media information

Plate 3: LB mix 1

Image

#

Date

Taken

Observations

1 5/3/12 Initial lawns setting up well, no GFP expression

2 5/4/12 No changes observed

3 5/7/12 White border along S.cerevisie= uracil exposure = dropped

plasmid4 5/9/12 GFP expression along the white line = E.coliexposed to arabinose

5 5/10/12 Increased GFP expression along interface of both strains

6 5/14/12 GFP expression and robust growth spreading back from border

7 5/15/12 GFP expression now along formerly white areas = yeast attacked

8 5/17/12 Further E.coligrowth, plate terminal


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Plate 7: SAB mix 1

Image

#

Date

Taken

Observations

1 5/3/12 Immediate contact observed, GFP expression at intersection

2 5/4/12 Rapid spread of E.colialong S.cerevisiaelawn; GFP expression

3 5/7/12 E.colispread across the top of the yeast lawn

4 5/9/12 Purple color fading from yeast growing under E.coli

5 5/10/12 E.coli growth continues, yeast has not spread at all

6 5/14/12 E.colicolonies stable, yeast continues to lose ground7 5/15/12 Darkening of the yeast colony, continued spread of E.coli

8 5/17/12 E.coliconsidered to have dominated plate, plate is terminal


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Plate 10: Serial Dilution of Hybrid culture

Image

#

Date

Taken

Observations

1 5/22/12 Colonies of E.coli and S.cerevisiaepresent from liquid culture; note

top left a white yeast colony.

2 5/24/12 No expression of GFP by E.coli,yeast colonies turning white

3 5/25/12 First visible evidence of GFP expression by E.coli

4 5/25/12 Closeup image of GFP expression by E.coli

5 5/29/12 GFP expression where E.colicolonies are adjacent to S.cerevisiae

6 5/31/12 E.coliseen growing into yeast colonies

Media recipes:

Media: LB Mix LB liquid mix SAB Mix SAB liquid mix

Sigma-Aldritch 5g LB .625g LB 6.00g SAB mix .75g SAB mix

4.00g Dextrose .5g Dextrose 2.00g Tryptone .25g Tryptone

3g Agar 3g Agar

200ml Water 25ml Water 200ml Water 25ml WaterAutoclave then: 400mg (2mg/ml)

L-arabinose50mgL-arabinose

400mg (2mg/ml)L-arabinose

50mgL-arabinose

Yield: 8 plates 25ml media 8 plates 25ml Media


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Literature Cited:

E. coli strain hb101 k-12.Bio-Rad Laboratories.Retrieved April 29, 2013, from

http://www.bio-rad.com/prd/en/US/LSR/SKU/166-0408EDU/E.-coli-Strain-HB101-K-12

pGLOTMPlasmid and GFP Kits.Bio-Rad Laboratories.Retrieved April 29, 2013 fromhttps://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-

b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kits

Sezonov, G., Joseleau-Petit, D., & D'Ari, R. (2007). Escherichia coli physiology in luria-bertani

broth .Journal of Bacteriology, 189(23), 87468749.

Keppler-Ross, S., Noffz, C., & Dean, N. (2008). A new purple fluorescent color marker for

genetic studies in saccharomyces cerevisiae and candida albicans.Genetics, 10(179), 705-

710.

Domingues, L., Lima, N., & Teixeira, J. (2000). Contamination of a high-cell-density continuous

bioreactor.Biotechnology and Bioengineering, 68(5), 584-587.

Verduyn, C., Postma, E., Scheffers , A., & Dijken, J. (1990). Physiology of saccharomycescerevisiae in anaerobic glucose-limited chemostat cultures.Journal of General

Microbiology, (136), 395-403.
http://www.bio-rad.com/prd/en/US/LSR/SKU/166-0408EDU/E.-coli-Strain-HB101-K-12https://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kitshttps://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kitshttps://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kitshttps://www.bio-rad.com/evportal/en/US/LSE/Category/f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed/pGLO-Plasmid-and-GFP-Kitshttp://www.bio-rad.com/prd/en/US/LSR/SKU/166-0408EDU/E.-coli-Strain-HB101-K-12

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