Difference between revisions of "Team:Yale/project"

Rationale for Project

Numerous genetic manipulation techniques have been developed for prokaryotes in the past decade, including zinc-finger nucleases, TALENs, SCALEs, CRISPR/Cas systems, and MAGE (Gaj et al. 2013, Lynch et al. 2007, Cong et al. 2013, Wang et al. 2009). However, these techniques have so far only been effective in a small handful of organisms beyond the model prokaryote Eschrichia coli. Despite their environmental significance and potential as producers of industrially-relevant small molecules, PCC7002, RT-CIAT, and many other non-model microbes lag far behind E. coli in terms of genome engineering technologies (Ramey et al. 2015). We reason that the development of a framework for the implementation of the most exciting genetic manipulation technologies into non-model organisms is necessary in order to maximize the positive potential which synthetic biology may have on industry and the environment. Back

CRISPR-Cas9 Systems

The search-and-modify tool of genome engineering

In the past several years, CRISPR-Cas systems have emerged as an extremely powerful gene perturbation and genome editing technique. Based upon the RNA-guided restriction endonuclease Cas9 from the microbial adaptive immunity system CRISPR (clustered regularly interspaced short palindromic repeats), these systems allow virtually any sequence within a cell's genome to be targeted (Hsu et al. 2014). The Cas9 protein relies on a 20-bp guide RNA (gRNA) sequence to base-pair directly with a DNA target. Once this occurs, Cas9 creates a double-stranded break (DSB) upstream of a sequence referred to as a protospacer-adjacent motif (PAM). The PAM sequence is unique for every organism which possesses a CRISPR system.

In a native CRISPR system, the gRNA is typically a sequence derived from a viral genome which is used to cleave foreign DNA if the cell becomes re-infected with the same virus. The CRISPR-Cas9 system's power as a genetic editing tool arises when the gRNA is used to target endogenous DNA. The Cas9 protein from the bacterium Streptococcus pyogenes is typically used, since its PAM sequence of NGG (where N = any nucleotide) allows the protein to create DSBs at a large number of loci (for instance, the sequence NGG occurs every 8 bp on average within the human genome) (Cong et al. 2013, Hsu et al. 2014).

Cas9-mediated DNA manipulation procedures hold promise in basic research, therapeutics, metabolic pathway engineering, and beyond. The Cas9 protein, guided by specific gRNA, can be coupled with a fluorescent reporter protein to visualize genomic structure in real-time. Cas9 under a light- or small molecule-inducible promoter, along with synthesized libraries of gRNA, can be transfected into organisms and induced to facilitate multiplex, genome-wide editing (Mali et al. 2013). The DSBs created by Cas9 can be repaired by homologous recombination-based (HR) methods or more error-prone non-homologous end joining (NHEJ), allowing for precise gene editing if HR is favored or nonspecific indel mutations if NHEJ is favored (Hsu et al. 2013). Back

Target Organism: Rhizobium tropici CIAT 899

A broadly symbiotic, nitrogen-fixing bacterium

Rhizobium tropici CIAT 899 is a promiscuous α-proteobacterium first isolated from a common-bean nodule in Colombia (Martinez-Romero et al. 1991). As a Rhizobium species, CIAT 899 can fix atmospheric nitrogen into a form that can be absorbed by plants, thus reintroduce nitrogen as an essential nutrient into the soil (New Mexico State University 2005). Rhizobium must nodulate plant roots in order to fix nitrogen, and although some Rhizobium species can only nodulate a limited number of legume species, CIAT 899 can establish nitrogen-fixing symbioses with a broad range of legume hosts (Hungria et al. 2000). CIAT 899 is more environmentally adaptable when compared to other Rhizobium species due to its tolerance of stressful conditions such as acidic soils and high temperatures (Graham et al. 1994). It has also shown resistance to various antibiotics, pesticides and fungicides (Bernal et al. 2004), all of which are properties that have led to CIAT 899's commercial use in inoculating the common bean Phaseolus vulgaris in South America and Africa (Ormeno-Orrillo et al. 2012). Because nitrogen-fixing legumes reduce reliance on synthetic fertilizers that are costly and ecologically damaging, scientists have recognized the role of Rhizobia in agricultural sustainability and ecological preservation (Balkan 2007).

R. tropici CIAT 899 is an ideal candidate for MAGE because it has a reasonably fast doubling time of < 6 hr (Morón et al. 2005) and its genome is fully sequenced (NCBI Taxonomy ID: 698761). CIAT 899 can produce a wide variety of Nod factor structures, which influence the range of symbiotic hosts (Morón et al. 2005). Although it is already used commercially to reintroduce soil nitrogen and has been very successful in increasing crop yields in some experimental plots, nitrogen-fixing legumes on average do not fix enough nitrogen to appreciably increase crop yield (Gilbert, 2012). There is potential for MAGE to further diversify the range of hosts that CIAT 899 require to fix nitrogen and increase the output of nitrogen from nitrogen-fixing symbioses. Back

Improving the Function of a Previously Existing BioBrick

We have improved the characterization of three previously existing BioBricks, namely three of the Anderson Promoters: BBa_J23100 (Anderson Strong), BBa_J23111 (Anderson Medium), BBa_J23114 (Anderson Weak).

We assembled these three promoters to citrine and the T7 terminator, and then ligated the constructs into two different vectors that could be transformed into R. tropici and S. meliloti. We measured expression using these promoters in E. coli, finding that bacA, lac, tac, and all three anderson promoters all drive expression to different extents. We then successfully transformed plasmids bearing promoter-citrine constructs into S. meliloti, finding successful expression for tac, Anderson Strong, and Anderson Medium.

We further assembled the Anderson Medium promoter to 5 different recombinases and confirmed that they were successfully transformed into S. meliloti. MAGE experiments are underway.

Our constructs have been confirmed by sequencing in pKT230, a broad-host range plasmid, and pPZP200, a plasmid that can be transformed into rhizobium and agrobacterium. The anderson weak-citrine and S-RSM4 single stranded binding protein constructs have been sequenced in the biobrick constructs, while sequencing for the other constructs is being done.

We also determined that BBa_K125000 does not have the advertised sequence, even as it does contain the spectinomycin marker and could also independently replicate in E. coli, as determined by our electroporation experiments.

References List