Difference between revisions of "Part:BBa K227007:Design"
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'''Method'''<br> | '''Method'''<br> | ||
| − | Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. <br><br> | + | Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. Oxygen tension was not able to be quantitatively measured due to the nature of the experiment.<br><br> |
For measurement of pucB/A expression from the puc promoter: <br> | For measurement of pucB/A expression from the puc promoter: <br> | ||
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. | 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. | ||
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===Source=== | ===Source=== | ||
| − | PCR | + | PCR applified from plasmid pRKCBC3 provided by Dr. C. Neil Hunter, University of Sheffield, Molecular Biology and Biotechnology. |
===References=== | ===References=== | ||
Latest revision as of 01:47, 22 October 2009
The puc promoter promotes transcription of the LH2 pucB/A genes naturally in Rhodobacter sphaeroides. The absorption spectra of a DBCOmega mutant (LH2 deficient) transformed with pRKCBC3 containing the puc promoter and pucB/A genes allows us to characterize the puc promoter under high and low oxygen conditions. More absorption of light at the LH2 spectra peaks normalized to culture OD corresponds with more transcription and vis versa.
Method
Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. Oxygen tension was not able to be quantitatively measured due to the nature of the experiment.
For measurement of pucB/A expression from the puc promoter:
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
puc promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 26
Illegal XhoI site found at 185 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 574
Illegal BsaI site found at 583
Design Notes
source: Braatsch et al. (2002)
Source
PCR applified from plasmid pRKCBC3 provided by Dr. C. Neil Hunter, University of Sheffield, Molecular Biology and Biotechnology.
References
Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in Rhodobacter sphaeroides." Molecular Microbiology. Vol. 45.3 (2002): 827-836.
Jager, Andreas et al. "The AppA and PpsR Proteins from Rhodobacter sphaeroides Can Establish a Redox-Dependent Signal Chain but Fail To Transmit Blue-Light Signals in Other Bacteria." Journal of Bacteriology. Vol 189.6 March 2009. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899404/pdf/1699-06.pdf]