Difference between revisions of "Part:BBa K227006:Design"

Revision as of 22:48, 21 October 2009

puc BA coding region of R. sphaeroides

Design Notes

puc A and puc B together create the LH2 antenna necessary for Rhodobacter sphaeroides to harvest light at wavelengths 800 nm and 850 nm. A common problem in bioreactors is the uneven distribution of light between cells nearest the light source and cells farther away from the light source. One way of combating this problem is to modify the antenna size of the organism. By truncating the light-harvesting antenna, cells at the exterior of a bioreactor waste less light through Non-Photochemical Quenching, thus allowing photons to penetrate deeper and reach cells in the interior of a bioreactor. This leads to an increase in photosynthetic efficiency and productivity for the entire culture.
This construct contains the natural RBS binding sites for R. sphaeroides.

LH2 Absorption Spectra
source: Walz et al. 1998
Characterization
pucB/A'
pucB/A are the genes that code for the beta and alpha subunits of the light harvesting complex LH2. Here, we measured the expression level of pucB/A from the puc promoter in the genome vs. on a low copy plasmid, pRKCBC3. This data allows us to utilize pucB/A as a reporter gene for expression from promoters, in complement to its role in cellular growth.
Method
Relative LH2 expression in R. sphaeroides 2.4.1, R. sphaeroides DBCΩ and R. sphaeroides DBCΩ pRKCBC3 grown anaerobically in the dark

Growth conditions: -Cultures grown in the dark at 34° C, shaking at 160 rpm - R. sphaeroides 2.4.1 -15 ml culture in polystyrene tube -15ml M22 was inoculated with 1ml inoculant with OD600nm = 0.270 - OD600nm (Volume) = 0.270 -Culture was allowed to grow 19hrs - R. sphaeroides DBCΩ -15 ml culture in polystyrene tube -15ml M22 was inoculated with 865ul inoculant with OD600nm = 0.312 - OD600nm (Volume) = 0.270 -Culture was allowed to grow 19hrs - R. sphaeroides DBCΩ pRKCBC3 -10 ml culture in polystyrene tube -10 ml M22 tet 5ug/ml was inoculated with loop of R. sphaeroides DBCΩ pRKCBC3 and capped with rubber stopper -Culture was allowed to grow for 3 days so that density was high enough to get a good reading

Analysis: Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength.
Results
File:02 puc B/A characterization graph.jpg
Conclusion
This data indicates that pucB/A can be utilized as a reporter gene as the LH2 absorption bands at 800 and 850 nm are not present in the LH2 deficient mutant DBComega. Furthermore, changes in the expression conditions of pucB/A are reflected on the absorption spectrum. In this case, expression is higher when the genes are expressed from the puc promoter within genomic DNA than on plasmid pRKCBC3, despite the fact that pRKCBC3 is maintained at 4-5 copies in a cell.

Source

PCR applified from plasmid pRKCBC3 provided by Dr. Neil Hunter.

References

Kiley, Patricia J.; and Kaplan, Samuel; "Cloning, DNA , and Expression of the Rhodobacter sphaeroides Light-Harvesting B800-850-a and B800-850-b Genes" Journal of Bacteriology. July 1987.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC212379/pdf/jbacter00197-0368.pdf]
Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" Journal of Molecular Biology Vol. 282 pp. 833-845