Part:BBa_K656012:Design
PowerCell
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 697
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 697
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 697
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 697
Illegal NgoMIV site found at 874 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 918
Illegal BsaI.rc site found at 2319
Design Notes
"Anabaena" separates the process of photosynthesis and nitrogen fixation spatially, into two cell types. Heterocysts are cells responsible for forming an anaerobic environment in which nitrogen fixation can occur. The more numerous cell type, vegetative cells, are photosynthetic and capable of producing excess sugars.
The promoter we have modeled our part after is present in the Photo-system I of Anabaena 7120, and therefore its activity is both constitutive and confined to vegetative cells. Our intent with this construct was to link the vegetative promoter to a process of sucrose secretion, by coding for a sucrose transporter from E. coli W. This was to confine sucrose secretion to only vegetative cells, as heterocysts are not producing sucrose through photosynthesis.
Our part is designed to secrete sucrose because other sugars like fructose and glucose are readily metabolized by the organism itself, and therefore less likely to be available for secretion. Our bigger picture goal would be to use the sucrose secreted by Anabaena to provide energy for other organisms like modified E. coli W.
The addition of GFPmut3b reflects our desire to easily characterize the functionality of the promoter, without necessarily characterization the activity of the sucrose transporter. In addition, cyanobacterial colonies require close to two weeks to grow up, and therefore to be dense enough to characterize sucrose transport, while GFP can be detected at much smaller amounts early on. We chose GFPmut3b because it's activity has been demonstrated in cyanobacteria.
Source
Anaebaena PCC 7120, E. coli W, part BBa_E0040
References
"Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products" http://aem.asm.org/cgi/content/abstract/76/11/3462
Archer, CT et al. The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics 12:9 (2011)
Lee, SY and Ho Nam Chang. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnology Letters 15:9 (1993) 971--974.
Lee JS, Lee SY and Sunwon Park. Fed-batch culture of Escherichia coli W by exponential feeding of sucrose as a carbon source. Biotechnology Techniques 11:1 (1997) 59-62.
Shukla, VB et al. Production of D(-)-lactate from sucrose and molasses. Biotechnology Letters 26 (2004) 689-693.
Zhang, X et al. Production of L-alanine by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 77 (2007) 355-366.