Team:Lethbridge/projectresearchdesign

Project

Research Design and Methods

1) Carbon fixation for fueling cell growth will be achieved through the incorporation of three genes into the genome of S. elongatus PCC7942 (Fig. 2). The E. coli galU gene, shown to increase sucrose production in S. elongatus by more than 30%7, will be incorporated to allow for high levels of sugar production. The Zymomonas mobilis invA and glf gene products will be used to cleave sucrose produced by S. elongatus into glucose and fructose and to export the sugars out of the cell. The 2011 Nevada iGEM team began characterizing the invA and glf genes for increased hexose sugar production in Synechocystis PCC680318 and submitted BioBricks for invA (BBa_K558006) and glf (BBa_K558005) to the parts registry. Glucose production by S. elongatus will be measured using established assays7, and the number of glucose-producing S. elongatus cells needed to sustain E. coli growth will be tested by spectrophotometry and cell viability assays. In parallel, the H. neapolitanus carboxysome operon, which has been successfully cloned into E. coli9, will be optimized and converted into BioBrick format. These BioBricks will be characterized for expression in E. coli by determining the ideal stoichiometry of each carboxysome protein and examining the carboxysome’s morphology and carbon fixation capabilities. Electron microscopy will be used to determine the structure of empty and cargo-loaded carboxysomes. Carbon fixation will be assessed in vitro and in vivo through established carbon fixation assays9. The sugars produced from carbon fixation will fuel E. coli cells engineered to produce (2) acetic acid and (3) rhamnolipid.

Figure 2. Glucose production by S. elongatus. Through carbon fixation, S. elongatus produces sucrose, which can be cleaved into fructose and glucose. The glucose transporter GLF will be used to export glucose out of the cell for use as an energy source for E. coli.

(2) Acetic acid breakdown of carbonate rock will be achieved through the production of acetic acid by E. coli. Upon treatment with acetic acid, the carbonate will become more porous and the production of CO2 will cause an increase in pressure, making the oil deposits more accessible. To produce large amounts of acetic acid, we will build a synthetic production construct with the E. coli acetogenesis enzymes PTA and ACK and the acetic acid efflux protein Aata from A. aceti13,14. We will optimize the expression of these enzymes and co-localize them with an RNA-protein scaffold19,20 to increase production and secretion of acetic acid from the cells (Fig. 3). Due to the multimeric nature of PTA and ACK, Gibson assembly will be used to rapidly assemble multiple BioBricks with different control elements (i.e. promoters and ribosomal binding sites) so to better control expression levels of the proteins used in the construct. We will characterize acetic acid production by using spectrophotometric methods to monitor cell growth in minimal media and performing pH titrations to determine acetic acid concentration in the media. Acetic acid production will be compared between cells expressing Aata and the scaffold system and cells expressing only Aata or the scaffold system. By co-localizing acetogenesis enzymes with Aata using the scaffold, the bacteria should produce larger amounts of acetic acid with limited growth effects. Although the common laboratory strain E. coli DH5α lacks certain acid tolerance mechanisms21, preliminary results show that the bacteria produce 340 mM acid on its own when grown under standard conditions. To improve acid tolerance, we will separately test the effectiveness of Aata at conferring acetic acid resistance by growing cultures of Aata-expressing cells in increasing concentrations of acetic acid to determine the inhibitory concentration of acetic acid. For industrial applications, the acetic acid secreted from the cells will be removed from the media and condensed for application to the carbonate rock, eliminating the need for direct application of bacteria to the oil deposits. The CO2 produced from the reaction of acetic acid and carbonate will be recycled by carbon fixation to continue fuelling the system.

Figure 3. Schematic of optimized acetic acid production and secretion in E. coli. Using an RNA-protein scaffold, the acetogenesis enzymes PTA and AcKA are co-localized with an acetic acid efflux protein (Aata) to increase production and secretion of acetic acid. The acetic acid will be collected from the growth media and condensed for application in CAB extraction.Scaffold assembly of acetic acid production enzymes. PTA and ACK will be overexpressed and joined by a scaffold to the membrane transporter Aata

(3) MEOR will be enhanced with the use of biosurfactants produced by E. coli. Rhamnolipid is a commonly studied biosurfactant produced by the enzymes RhlA and RhlB in P. aeruginosa, which have been successfully cloned into E. coli16,22. The 2010 and 2011 Panama iGEM teams have worked on creating BioBricks for the P. aeruginosa enzymes needed for rhamnolipid production23. We will examine the commercial viability of biologically producing rhamnolipid for EOR by first optimizing the expression of RhlA and RhlB in E. coli and quantifying the extent of rhamnolipid production through established assays16. The ability of rhamnolipid to act as a biosurfactant in the presence and absence of acetic acid will be tested to assess the effectiveness of rhamnolipid for EOR.

(4) Environmental safety will be ensured by utilizing natural methods for degrading genomic DNA and proteins of our chassis. We will use a constitutively expressing promoter (J023100) linked to a high expressing (34%) ribosomal binding sites (B0034) to control expression of the proteins YF1 and FixJ. When exposed to light at 470 nm, YF1 and FixJ will dimerize around the promoter region pFixK, and prevent the transcription of ClpXP, ClpP and RecA24. In the absence of light at 470 nm, the expression of the protease ClpP, the protease specific enhancing factor ClpXP, and the recombinase RecA will no longer be repressed. These proteins act to degrade essential cellular proteins and DNA, resulting in cell death. ClpXP is able to degrade proteins through identification of a protein tag conserved across species so will be a useful module in both of our chasses25. Additionally, S. elongatus can use 470 nm light for growth, making this an ideal expression system for CAB extraction. The expression system will be tested by monitoring cell viability in the presence of varying wavelengths of light, as well as the length of time in the absence of 470 nm light needed to effectively kill the cells. Since bioreactors can be used for CAB extraction, growth conditions can be tightly controlled to prevent degradation while the bacteria are needed to produce CAB products. However, daily light cycles will ensure that any bacteria removed from the bioreactor will be rendered inert and non-threatening to the environment.

Figure 4. Light-repressible kill switch module. Constitutive expression of heterodimer pair, YF1 and FixK, when exposed to light at 470 nm will prevent expression of the kill switch modules. When the bacteria are not exposed to 470 nm light, YF1 and FixK will not be expressed, which will allow for expression of the recombinase RecA and the proteases ClpP and ClpXP, which will destroy genomic DNA and essential proteins, rendering the cell inert.