Team:Brown-Stanford/PowerCell/NutrientSecretion

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Brown-Stanford
iGEM

Nutrient Secretion

PowerCell is the generator that will power all of the BioTools in a Martian settlement. PowerCell will secrete nutrients for use by other biological tools as both a source of energy and raw materials to construct useful products.

Brown-Stanford Battery.png

Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are carbon sources (sugars) and nitrogenous nutrients (ammonia). In a Martian settlement, PowerCell will harness the energy of the sun to harvest these nutrients directly from the atmosphere and provide them to other biological tools.

This summer, we tackled only the half involving sugar secretion. This was partially to due to the time constraints of a summer project, and partially because there is evidence that ammonia secretion occurs in cyanobacteria by means of passive diffusion without the need for genetic tinkering (Ammonia translocation in cyanobacteria). More research needs to be done to determine if this level of diffusion is sufficient to sustain other biological tools. In any case, we designed our system on a platform with the capability to fix atmospheric nitrogen, so that this avenue is open for exploration in the future.

Choice of Host Strain

Anabaena 7120 is a filamentous bacterium capable of both photosynthesis and nitrogen fixation. Anabaena forms two cells types along the filament: vegetative cells, which perform photosynthesis, and heterocysts, which fix nitrogen. The nutrients are then shared up and down the chain. In this image, the heterocysts are the slightly larger oblong cells spaced approximately every eighth cell.

For our host, we chose a diazotropic cyanobacterium, Anabaena 7120. Anabaena 7120 has several special characteristics that make it an advantageous host species. First, it can fix its own nitrogen into biologically usable ammonium directly from the atmosphere, and second, it can do this at any time of day. The process of nitrogen fixation is detrimentally affected by the presence of even a small amount of oxygen. For this reason, most diazotrophs separate the processes of photosynthesis and nitrogen fixation temporally – that is, they photosynthesize in the day and fix nitrogen at night. Anabaena 7120 has a different way around this problem. It forms long filaments of cells containing two different cell types, normal (vegetative) cells, and heterocysts. Heterocysts form thick cell walls and house very anaerobic environments; they are where nitrogen fixation takes place. Normal (vegetative) cells carry on with photosynthesis. The two cell types then share nutrients up and down the filament, thus providing each cell with all the nutrients it needs.

The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium S. elongatus.

Sugar secretion Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Niederholtmeyer et al.

In a nutshell, S. elongatus were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.

At the Fifth International Conference on Synthetic Biology, SB5.0, we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did, although the choice does have some ramifications.

Because E. coli is the a primary host species for genetic modification, it is likely that many biological tools for space exploration will be designed in E. coli. The most common laboratory strains of E. coli , TOP10 and K12, cannot directly metabolize sucrose. For this reason, they cannot make use of PowerCell's output directly. In order to utilize this output, sucrose will need to be broken down into glucose and fructose , or biological tools that intend to utilize PowerCell should be hosted by a strain that can directly metabolize sucrose. E. coli W is a safe and easily transformable laboratory strain with this ability, and, as outlined in The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli, may confer some other advantages to biological tools to be used in space, including including fast growth, stress tolerance, growth to high cell densities. Furthermore, there is some indication that sucrose as a feedstock can confer other benefits, such as oxidative, heat, and acid stress (Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for l-threonine production).

PowerCell Contruct Design

We wanted to isolate sucrose secretion to just vegetative cells, as heterocysts do not participate in production of sucrose through photosynthesis. A similar cell-type-specific gene expression was was achieved in Construction and use of GFP reporter vectors for analysis of cell-type-specific gene expression in Nostoc punctiforme. Arguetta et al. confined expression of a fluorescent marker to only vegetative cells in a similar filamentous diazotrophic cyanobacterium, Nostoc Punctiforme. They placed the GFP gene behind a promoter present in the Photosystem I of Nostoc punctiforme, psaC. This way, GFP expression was only turned on in photosynthesizing cells, and not in heterocysts. They did not know the exact sequence of the psaC promoter, so they just took the 400 base pairs upstream of the psaC gene as a unit containing the promoter. We have done similarly to isolate the psaC promoter from Anabaena 7120. Below is an image showing cell-type-specific GFP expression achieved in the paper.


Cell-type-specific GFP expression in Nostoc Punctiforme. Arguetta et al.


We placed a sucrose symporter gene, cscB, behind our Anabaena 7120 psaC promoter in order to confine sucrose secretion to only vegetative cells. Registry standard RBSes and terminators were used for ease of assembly and compatibility. Below is a diagram of our sucrose secretion device. A discussion of the inner workings of the cscB gene can be found in Active Transport by the CscB Permease in Escherichia coli K-12.


Brown-Stanford cscB.jpg


We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria (Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology).


Brown-Stanford GFP.jpg


Tranformation of our construct into Anabaena

Our first trials were performed with the helper plasmid pDS4101, the conjugative plasmid pRL443 and the cargo plasmid pRL25. pDS4101 does not possess the methyltransferases which can epigenetically protect the incoming DNA from restriction, and so can only be used to transfer plasmids lacking restriction sites, such as pRL25, as a proof of concept.

Towards the end of the summer we were able to obtain the helper plasmid pRL623 containing the three methyltransferases which together offer complete protection from restriction inside Anabaena. There was an added obstacle with this new route, however: pRL623 could only be carried against certain E. coli genetic backgrounds lacking methyl-restricting enzymes, in order to prevent restriction of the host genome. This meant that transfer of the helper plasmid into the cargo plasmid strain before conjugation into Anabaena would result in the suicide of that cargo strain, and no transformation. The solution to this was transformation by more traditional means of the cargo plasmid into the strain already carrying the helper plasmid; the cargo could then be delivered into Anabaena without transfer of the dangerous helper plasmid, and the helper plasmid had its opportunity to protectively methylate the cargo prior to transformation.

We are sincerely grateful to Jeff Elhai, Peter Wolk, James Golden and Norman Wang for their crucial help and guidance.


Triparental mating: Our desired construct (from the donor strain) and a helper plasmid are inserted into a helper strain. The helper strain and conjugative strain are spotted with the recipient Anabaena for the three-parent mating

Utilizing sucrose

E. coli W

An essential aspect of the PowerCell project lies in microorganisms being able to survive off the nutrients being secreted by our genetically engineered Anabaena. Our carbon source, sucrose, is a disaccharide that is not necessarily able to be metabolized by all species.

Here our project intersects with current trends of bioproduction on Earth; sucrose is a cheap and easily obtained sugar, so there is interest in engineerable strains of microorganisms able to survive on it as a sole carbon source.

Sugarcane is an extremely low-cost source of bulk sucrose used in production [1]

We came across research suggesting that E. coli W is one such promising strain. It is one of the few strains of non-pathogenic E. coli able to metabolize sucrose, has good tolerance for environmental stresses, and grows rapidly. (Archer 2011) There have been papers describing the process of adapting E. coli W as a bioreactor organism, and methods of growing them to high density and productivity using fed-batch cultures. (Lee 1993, Lee 1997).

In fact, the strain has already been used to produce a number of useful products. These include D(-)-lactate, a precursor to the formation of certain biodegradable plastic polymers, and L-alanine, a food additive and nutritional supplement. (Shukla 2004, Zhang 2007) With these examples, it is not difficult to imagine the production role of E. coli W in our Martian colony.

Experiment

In the application of our project, PowerCell would be grown alongside and supporting a productive microorganism strain such as E. coli W. We were thus interested in seeing whether E. coli W could grow in the conditions generated by PowerCell. This meant culturing on minimal BG-11 media with added sucrose to simulate secretion by Anabaena.

Based on literature (Niederholtmeyer, et al,2010) and correspondence with Mr. Ducat, we predicted that our Anabaena culture would produce no more than 8mM of sucrose. It would be difficult to support substantial E. coli growth on such low levels without a means of concentrating sugar via in the bioreactor design.

Initially we attempted to compare growth on BG-11 +sucrose with liquid cultures. However, multiple attempts yielded no appreciable change in O.D. after several days of incubation. We suspect that this was because E. coli growth was too small to detect via spectrophotometer.

Next, we moved to agar plates as a more sensitive means of detecting cell growth. E. coli W with Amp resistance were grown, washed and resuspended in PBS to eliminate residual LB media, and streaked them on BG-11 Amp+ plates with varying concentrations of sugar (5mM, 10mM, 30mM, 60mM, and 80mM).


Growth of our E. coli W varies by amount of sucrose (there was no 200ul plate for 80mM sucrose)
E. coli W colonies on BG-11 agar + sucrose


The cells were incubated at 37C and observed regularly before colonies appeared on the fifth day. These data suggest that, with the sugar secreted by PowerCell expressing cscB and no additional metabolic engineering, it is possible to support another organism.



References

Archer, CT et al. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics 12:9 (2011)

Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.

Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.

Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.

Zhang, X et al. Production of L-alanine by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 77 (2007) 355-366.

H. Niederholtmeyer, et al. Engineering cyanobacteria to synthesize and export hydrophilic products. Appl. Environ. Microbiol., 76 (2010), pp. 3462–3466.