Team:Brown-Stanford/PowerCell/NutrientSecretion

Nutrient Secretion

PowerCell is the generator that will power all of the BioTools in a Martian settlement.

[picture of bio battery]

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 sugars (carbon sources) and nitrogenous nutrients. In a Martian settlement, PowerCell will harness the energy of the sun and matter from the surrounding atmosphere to provide both these necessities.

CARBON SECRETION: WHY WE CHOSE SUCROSE (SILVER LAB, DUCAT CORRESPONDENCE, CELLULAR BENEFITS OF SUCROSE)

NITROGEN SECRETION: WHY WE DIDN'T FOCUS; EVIDENCE OF LEAKAGE?

CONSTRUCT SCHEMATIC, EXPLAIN EACH COMPONENT: ANABAENA PROMOTER: HOW DID WE DERIVE THIS METHOD OF LIMITING EXPRESSION TO VEGETATIVE (CITE PAPER; SHOW PRETTY PICTURE OF LOCALIZED GFP EXPRESSION)

CSCB; EXPLAIN HOW SUCROSE SYMPORTER WORKS, (CITE PAPER WHERE IT IS DESCRIBED IN E COLI W)

GFP REPORTER CONSTRUCT SCHEMATIC; WHAT IT IS THERE FOR, HOW WE EXPECT IT TO WORK

GFPMUT3B; WHY REGULAR GFP WON'T WORK (CITE PAPER WHERE MUT3B IS VISIBLE OVER BACKGROUND PIGMENT LEVELS)

TRANSFORMATION THIS IS HOW WE TRIED/PLAN TO GET OUR CONSTRUCT INTO OUR ANABAENA TRIPARENTAL MATING DESCRIPTION, RESTRICTION ENZYMES, HELPER PLASMIDS GALORE -MENTION CORRESPONDENCE WITH ELHAI, WOLK

This summer, we tackled only the half involving sugar secretion. We built our system, however, on a platform that can support nitrogen fixation and secretion if someone develops it in the future.

[Anabaena picture]

For our host, we chose a diazotrophic (link to definition) 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 (title, link). 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.

[Silver invA diagram]

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, (link to outreach page) 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.

Below is a diagram of our sucrose secretion device. We placed the sucrose symporter gene, cscB, behind a promoter present in the Photo-system I of Anabaena 7120. This was done to confine sucrose secretion to only vegetative cells, as heterocysts are not producing sucrose through photosynthesis.

[Ana-cscB diagram]

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.

[Ana-GFP diag]

[Ana-cscB-GFP diag]

One major challenge was transforming our constructs into Anabaena 7120. Anabeana has several restriction enzymes, AvaI, II, and III, which prove problematic for routine transformations into the species. The method required for such a transformation was developed by Elhai and Wolk (cite paper). It requires a conjugal mating of the target cyanobacterium with two strains of E. coli, containing the plasmid of interest and a conjugal transfer plasmid, respectively.

Utilizing sucrose