Improved procedures for Genetic Transformation of Rhodobacter capsulatus

Goals and Motivation

Rhodobacter capsulatus has the potential to be very useful for production of various compounds, including botrycoccene (a biogasoline precursor) and squalene (a steroid precursor originally harvested from shark liver oil) because of its ability to be grown to high densities on CO2 and H2. However, to make better advantage of this biosynthetic capability, an efficient method for the genetic engineering of R. capsulatus is needed. Due to a highly active restriction system, insertion of heterologous DNA into R. capsulatus through two commonly used methods, transconjugation and electroporation, is very difficult and these processes have not been thoroughly optimized. Therefore, the purpose of the following experiments was to improve the current transformation protocols so that genes encoding for botrycoccene and squalene can be expressed and optimized in R. capsulatus.

Serena Xu, John Pisciotta, Wayne Curtis

Department of Chemical Engineering, The Pennsylvania State University, University Park, PA

Electroporation vs. Conjugation

Vector used for heterologous gene expression in R. capsulatus3

Transconjugation
Through experimentation, it was found that in ratios ranging from 10:1 to 50:1 R. capsulatus:E. coli S17, the ratio that gave optimal results was 50:1. It was also observed that the vector pBBR gave more promising results than pIND, possibly due to the poor anitibiotic resistance of pIND; to test this theory anitibiotic resistance of pBBR was cloned into pIND by Chaoxuan Li to create new pIND vectors. When old pInd vectors were attempted to be conjugated into R. capsulatus, between 0 and 2 exoconjugants resulted. New pInd vectors result in between 600 and 6000 exconjugants, an efficiency of between 104 to105 g-1 plasmid DNA resulted.

Electroporation: application of an electric shock causes temporary pores to form in the cell membrane, through which DNA and other molecules pass. When the membrane reseals, the DNA is trapped inside. Conjugation:
Electroporation creates pores in the cell membrane4

1) mating

Mixing improves conjugation efficiency of R. capsulatus

conjugants/g plasmid DNA
8000 7000 6000 5000 4000 3000 2000 1000 0

2) Selection plating (kills E. coli and selects for R. capsulatus with plasmid 3) Restreaking confirms conjugant The ability to receive and donate DNA is natural to many species of bacteria, as this ability can be very helpful for survival, for example, by transferring antibiotic resistance.

Mixing improves efficiency of conjugation5

BR BC3- BR BC3- BR BC3- BR BC33 3 4 4 mixed mixed

500:1 R. capsulatus:E. coli with mixing maximizes efficiency of conjugation

4000 conjugants/g plasmid DNA 3500 3000 2500 2000 1500 1000 500

Botrycoccus braunii2

Genes similar to those in the algae Botryococcus braunii that will hopefully help used to produce botryococcene and squalene and their precursors, will be genetically engineered into R. capsulatus

Electroporation
Electroporation of heterologous DNA into R. capsulatus has not yet been found possible. However, we have found electroporation of homologous DNA possible with a small broad host-range vector, pBBR1MCS-2 at a low rate of about 1x103g-1 plasmid DNA, indicating that restriction enzymes are digesting the heterologous DNA. Since conjugation of R. capsulatus is now working reliably, more attention will be focused on first optimizing electroporation of homologous DNA. The optimized procedure can then be to test methods to circumvent the restriction system, such as unmethylated DNA, applying various stresses to R. capsulatus, or engineering R. capsulatus methylases into E. coli.

0 50 (mid log phase):1 250 (mid log 250 500 (mid log phase):1 (stationary):1 phase):1
Ratio R. capsulatus (age):E. coli (cells)

500 (mid log phase):1, mixed

Map of triterpene production from precursor mevalonate with genes encoding each step

References
1Texas

AgriLife Research photo. 2Kovach, Michael E., Philip H. Elzer, Steven Hill, Gregory T. Robertson, Michael A. Farris, Martin Roop, and Kenneth M. Peterson. "Four New Derivatives of the Broad-host-range Cloning Vector PBBR1MCS, Carrying Different Antibiotic-resistance Cassettes." Gene 166.1 (1995): 175-76. 3http://www.inovio.com/images/IMG_electroporation.gif 4Del Campo, Irene, Raul Ruiz, Ana Cueves, Carlos Revilla, Luis Vielva, and Fernando De La Cruz. "Determination of Conjugation Rates on Solid Surfaces." Plasmid 67.2 (2012): 174-82.

Acknowledgements
Funding for this summer research experience was provided by an Endowment to the Penn State Chemical Engineering Department for work in the area of Biomolecular Engineering. Training was provided by Chaoxuan Li. Funding for the experiments was provided by ARPA-E grant # DEAR0000092 for thedevelopment of Rhodobacter as a versatile platform for fuels production.

Ratios higher than those previously tested were tried, with mixing at the highest ratio (mixing is more effective at higher ratios). The highest ratio with mixing was found to result in the greatest efficiency of DNA transfer. Collaborators had found contradicting evidence of whether R. capsulatus in an earlier or later growth stage result in better conjugation. I found that the difference is not significant, but using older R. capsulatus makes other factors easier to control.