Team:Nevada/Project/Co-Cult

Introduction

      Co-culturing multiple bacterial strains is inherently complicated. The two species must co-exist while at the same time competing for resources. Over time the growth rate of two co-cultured species will establish equilibrium. However, conditions will not be optimal for either individual species. In our case, we want to grow the E. coli in a culture that is being provided with glucose from the cyanobacterium, Synechocystis PCC6803. It is clear that E. coli growth will be limited by the productivity of the cyanobacterium. Therefore, we need to develop a system that will optimize Synechocystis growth. One of the major considerations in cyanobacteria growth is the availability of sufficient light to optimize rates of photosynthesis. This is particularly important in our system since we are also depending on photosynthesis for the production of glucose to feed E. coli. While it is possible to grow E. coli and cyanobacteria in the same culture vessel (Niederholtmeyer et al., 2010) Applied and Environmental Microbiology 76: 3462-3466), the photosynthetic efficiency of the system will be limited by light blockage caused by E. coli. For this reason, we have developed an apparatus that physically partitions the two bacterial species from each other, while still allowing for the free exchange of growth medium. In this way, we can ensure high photosynthetic rates for Synechocystis and total accessibility by E. coli to the cyanobacterial produced glucose.

Choice of Growth Medium

      BG-11 must be used for culturing Synechocystis. However, this medium does not provide adequate nourishment to E. coli. Thus, this project must determine what additives to BG-11 will foster growth of both organisms. The supplemented media must allow for growth of E. coli comparable to LB but must not inhibit Synechocystis with toxic levels of solutes.

      Initial tests will determine if BG-11 has an inherently sufficient nitrogen supply for E. coli. Previous research has shown that NH₄⁺ is more effective than nitrates and nitrites when used by E. coli as a nitrogen source (Strevett, K.A., and Chen, G., 2003). Therefor, testing will be done with the growth of NEB 10-ß cells in BG-11 with NH₄Cl as an additional source of nitrogen. The growth in this media will be compared with that of BG-11 alone. It must then be shown that Synechocystis PCC6803 can tolerate the levels of ammonium chloride required. If ammonium chloride cannot be used, nitrites and nitrates may then be used as a backup option.

      Once a source of nitrogen has been found, experiments will be done to find a standard curve. This curve will give us a baseline we can compare with when determining if our E. coli is being successfully fed by glucose produced in cyanobacteria. The ideal media will produce an optical density of 1.00, at 600 nm, after 24 hours. When glucose is added to this media, we should see a greater extent of growth, giving a positive result for co-cultivation.

Results of Growth Curves

Growth in BG-11 + NH₄Cl

      It was quickly found that 10-ß growth was limited in BG-11 due to one or more auxotrophies. Upon contacting NEB tech support, it was confirmed that 10-ß cells are deficient in leucine synthesis. Thus preventing them from growing in BG-11 that is not supplemented with this amino acid. However, further testing with BG-11 and leucine indicated that 10-ß still could not grow significantly in this media.

      Testing was then done with other cell lines, in an attempt to find a cell line that was not inhibited by amino acid auxotrophies. BL-21 cells were first used, with no luck. NEB tech support was again contacted and asked for a cell line that was not deficient for any amino acid synthesis. NEB recommended Express I^q cells, claiming that they were not auxotrophic and would be ideal for expression studies using the pTRC promoter. Unfortunately, it was found that even I^q cells were not capable of significant growth in media lacking amino acid supplements.

Growth in BG-11 + Amino Acids

      Upon finding that none of the cell lines available to the team were capable of growing in BG-11 supplemented with ammonium chloride as the only nitrogen source, sources of amino acids were looked at. The first of these sources was tryptone, a trypsin digest of casein providing all amino acids. Our studies showed that a concentration of 0.5% w/v tryptone produced our desired standard curve, with an average OD at 600 nm of 0.992 (n=2), 25 hours after inoculation with an overnight culture. This experiment was done using 10-ß cells. Readings on the contents of tryptone showed that it contained unacceptable amounts of sugars (Biotech Solabia Group). This would interfere with our studies into the effects of adding glucose to our media.

      The next source of amino acids tested was casamino acids, another product of casein. However, casaminos provide greater amounts of free amino acids and small peptides and do not contain notable quantities of sugars (BD Difco). Our experiments produced the desired standard curve with a concentration of 0.25% w/v casaminos, in the case of 10-ß, and 0.20% w/v for I^q.

Detection of Glucose

      With standard curves found for both 10-ß and I^q, using casaminos, experiments were done to determine if we could induce greater growth with the addition of glucose. Our initial experiment was with 10-ß and glucose was added in 10, 25 and 50 mM amounts. The data showed that 10-ß responded negatively to added glucose, giving 600 nm ODs of greater than 35% less than our standard curve at all concentrations.

      An identical experiment was performed with I^q cells. In this case, glucose increased growth by up to 38% (at 25 mM glucose) over the standard curve after 25 hours. With a positive result, the experiment was repeated in triplicate. The results confirmed those of the initial experiment. In the presence of 10 mM glucose, the average OD at 600 nm, after 24 hours, was 67.9% (n=3) greater than our standard curve.

      Having confirmed that I^q cells respond positively to glucose in BG-11 media with casamino acids, we wanted to look at the levels of glucose we would need to feed our E. coli. Other researchers have shown that Synechocystis PCC6803 agp knockouts are capable of producing 10 mM sucrose (Zhao, N., et al. 2003). Another group has shown that introducing invA to cyanobacteria can convert nearly all cellular sucrose to fructose and glucose (Niederholtmeyer et al., 2010). On the notion that 10 mM sucrose can be produced by knocking out agp and all sucrose can be converted to glucose, it was hypothesized that our maximum glucose output from Synechocystis would be 10 mM. Our previous experiments had shown that there was no noticeable difference in growth increases between 10, 25 and 50 mM glucose, with 10 mM actually showing the greatest increases. If we could detect even lower amounts than our hypothesized maximum, we would be ready to begin co-culturing. An experiment was done, in triplicate, in which glucose was tested in BG-11 in the following decreasing concentrations: 10, 5.0, 2.5, 1.0, 0.50 mM. Even at concentrations as low as 2.5 mM I^q cells showed increases in growth over the standard curve of up to 60%. Even at concentrations of 500 µM, growth was increased more than 10% greater. With confirmation that such small amounts of glucose can successfully nourish E. coli, it was time to begin testing the co-culturing apparatus.

Apparatus Components

Culture Vessels
    The actual chambers are made from modified chromatography columns. They are Pyrex glass tubes with removable plastics caps. Each chamber has a double seal to prevent leaking. Both of these are supported by a ring stand with clamps.

Transfer Pump
      One main thing required to have an effective apparatus is a pump to transfer one bacteria solution to the other chamber into a permeable membrane. It needs to be able to transfer enough volume to fill the dialysis tube completely but not create too much pressure that the tubing would burst. An external pump was chosen to reduce the number of components that would touch the bacteria and lesson the chances of contamination. It also reduces the amount of parts to be cleaned. On our apparatus the E. coli is gravity fed to the transfer pump inlet, then pumped through clear vinyl tubing to the dialysis tube and returned back to the E. coli chamber. It pumps approximately 25 gallons/hr.

Semipermeable Membrane
      The dialysis tubing allows regulated bacteria interaction, meaning glucose from the Cyanobacteria can transfer over to the E. coli but the two bacteria never contact. Something was needed to give the dialysis tubing rigidity and also allow effective flow of E. coli through the inside of the tubing. The dialysis tubing also needed something to seal the ends to prevent contamination. A borosilicate tube was taken and had bubbled ends installed for the sealing surface to the dialysis tubing. The glass tubing then had small holes placed in it with a wall in the middle. This made one end an inlet where the media would enter the glass tube flow out of the small holes into the dialysis tubing filling it on its way to the other end. From there it would exit through the other set of small holes and return to the vinyl tubing and the second chamber. To seal the dialysis tubing to the glass a piece of shrink tube was placed over the dialysis tubing compressing it against the glass. Next a rubber o-ring was placed over the shrink tube to provide an extra seal.

Oxygenation
      For proper growth, each chamber needs a constant flow of oxygen. A four-channel variable aquarium oxygen pump, inside the apparatus base, oxygenates the Cyanobacteria and E. coli. Each chamber is fed from the bottom through two lines, check valves were installed to prevent the bacteria from draining down the tubing into the oxygen pump. On the top of the base is the adjustment knob to control the oxygen flow. Hand blown glass bubblers, made from borosilicate glass, were attached to the top of each chamber to allow proper venting without releasing the bacteria out.

Illumination
     In order for photosynthesis to take place some sort of artificial light is necessary. Two T5 14W fluorescent bulbs, each connected to a slider that allows them to be independently adjusted to vary the amount of light as needed in the Cyanobacteria chamber.

Controller
      The base is constructed of 6 panels of 6061 aluminum tig(GTAW) welded together. Its dimensions are approximately sixteen inches long, fourteen inches wide, and three inches tall. All electrical switches and wiring are inside with the oxygen pump and rings stand base to keep bacteria out and make it easier to clean.