Team:Hawaii/Project

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Overall Project

A BioBrick toolkit for cyanobacteria

We aim to extend the current BioBrick registry to a greater range of organisms, including cyanobacteria. Cyanobacteria are studied for their ability to produce useful compounds, including biofuels and biopolymers. These "little green factories" require only salts, light, water, and carbon dioxide for photoautotrophic growth. A cyanobacterial "toolkit" would enhance our ability to utilize this system.

We designed:

1) mobilizable broad-host range BioBrick vectors derived from RSF1010,

2) a cassette for protein secretion from Synechocystis sp. PCC 6803, and

3) a nitrate-inducible cyanobacterial promoter BioBrick.

Our toolkit was designed for conjugative gene transfer from Escherichia coli to Synechocystis to achieve the controlled production and recovery of bioproducts, demonstrable by induced secretion of green fluorescent protein. Though our parts were targeted for work in cyanobacteria, they may be compatible with other Gram-negative systems including Agrobacterium, which is capable of plant transformation.

Project Details

Part A: Mobilizable Broad-Host-Range Plasmid

RSF1010 is a naturally occurring broad-host-range plasmid capable of conjugative transfer and stable replication due to the presence of mob genes with an associated origin of transfer (oriT) and rep genes with an associated origin of vegetative replication (oriV), respectively. We aim to compartmentalize a derivative of the RSF1010 plasmid, namely pRL1383a, into BioBricks. The resulting BioBricks can be inserted into a BioBrick base vector to create a plasmid that transfers genetic elements via conjugation. (read more...)

Part B: Cyanobacterial protein secretion system

Photosynthetic cyanobacteria provide the opportunity for autotrophic production of practically any biomolecule. The ability to extract engineered biomolecules would make this bacterium a renewable, nearly self-sustaining "factory"- a potentially valuable tool in bioengineering. For Part B, we will create BioBricks encoding naturally occurring signal peptides that can be combined with a protein coding sequence in order to express the protein of interest extracellularly. (read more...)

Materials, Methods, and Results

Part A: Mobilizable Broad-Host-Range Plasmid

The aada BioBrick containing the lac promoter and rbs and the origin of transfer have been successfully constructed. The aada BioBrick was confirmed by sequencing and was tested in E.coli. The construct was shown to confer resistance to streptomycin and spectinomycin. The origin of transfer was confirmed by sequencing and will be tested in E. coli using the construct with oriT inserted into the lacZ BioBrick (J33207) plasmid.

The Broad-Host-Range plasmid is still under construction. Currently the complete set of parts -- replication proteins, the origin of vegetative replication, the aadA gene and the origin of transfer are in pSB1A3 plasmid and are currently being tested. We plan to place this construct in the BioBrick Base Vector, though as it is in pSB1A3, all of the parts necessary to verify that the plasmid is transferred through conjugation, and is autonomously replicating in Synechocystis are present. (Details...)

Part B: Expression system for Synechocystis sp. PCC 6803

The nitrate inducible nir promoter and the pilA and slr2016 secretion signal sequences were successfully constructed from synthesized oligonucleotide fragments. GFP, BBa_E0040, was converted into a fusion brick through site-directed mutagenesis. Though ligation of pilA to any BioBrick part was unsuccessful, we were able to ligate slr2016 to GFP fusion to eventually create a constitutive GFP secretion device under the control of a lac promoter. E. coli strain DH5α transformed with this device was observed to glow green. However, GFP secretion was not observed since the signal sequence is specific to Synechocystis sp. PCC 6803.

The mobilizable broad host range plasmid, pRL1383a, was successfully converted into a BioBrick plasmid. The original pRL1383a MCS was replaced by the BioBrick part J33207 and the full pSB1A2 flanking regions. J33207, a lac promoter-lacZ construct, allows for blue-white selection of transformants. However, when J33207 was inserted into pRL1383a, no blue colonies were observed though the insert was confirmed by sequencing. Sequencing revealed a single base pair transversion in the CAP binding site of the lac promoter. It is unknown whether this mutation causes the lost of blue/white screening capabilities, or if the plasmid itself is affecting transcription or translation of the lacZ gene.

The constitutive secretion device has been placed in the converted pRL1383a plasmid and is currently being conjugated into Synechocystis sp. PCC 6803.

The intended nitrate-inducible GFP secretion device and controls are in the process of being constructed. When completed, these devices will also be conjugated into Synechocystis sp. PCC 6803 for testing. (Details...)

Implications and future plans

The availability of an additional broad-host range plasmid allows for testing of additional BioBrick constructs in other gram-negative organisms transformable by pRL1383a. Genes coding for proteins of industrial value can be converted into fusion brick format and ligated to either of the signal sequence bricks we have created. The resulting construct can be introduced into the organism of interest using the plasmid K125000 or K125010. Synechocystis transformants should be capable of secreting the protein into culture media, optimizing production since multiple yields can be collected over time from the living cell culture.

Further testing is still necessary to fully characterize the plasmids we have produced. We also need to work out the protocol for blue-white selection in DH5α using K125000. Though the correct BioBrick part is included in the plasmid, colonies do not appear blue. In addition, transformed E.coli colonies grow slowly and do not appear uniform in size.

Since the plasmid pRL1383a is a low copy number plasmid, it may be useful in the future to supplement the plasmid with an inducible high copy number origin of replication. An appropriate promoter could be selected so the plasmid replicates as a high copy number plasmid when placed in E.coli but reverts to a low copy number when transferred to cyanobacteria or other gram-negative organisms.

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]