The Registry of Standard Biological Parts is a library of DNA sequences combined with online characterization resources. The Registry has created a standard protocol for making segments of DNA compatible with all other segments of DNA, regardless of order or size. BioBrick is the term given to such segments of DNA, a term alluding to the fact that any number of bricks may be combined in any order to produce complex, unique systems. This is accomplished by standardizing the restriction enzymes used to surround BioBricks, as well as the plasmids used to transform them. For a graphical representation of of the process, please click here. A powerful online database provides information and characterization of all of the BioBricks in the Registry and uses the wiki format (the same one used in wikipedia) which encourages others to edit content directly on the page. Below are list of parts that were used/created for this project.
Parts used to characterize and build our final project
Component | Part/Accession # | Base Pairs | Plasmid | Resistance | Well |
RBS | BBa_B0034 | 12 | pSB1A2 | Ampicillin | plate 1, 2M |
RBS | BBa_B0032 | 13 | pSB1A2 | Ampicillin | plate 1, 2I |
RBS | BBa_B0030 | 15 | pSB1A2 | Ampicillin | plate 1, 1H |
Cph8 | BBa_I15010 | 2,238 | pSB2K3 | Kanamycin | N/A |
RFP | BBa_J04051 | 720 | N/A | N/A | N/A |
OmpR (E. coli) | BBa_K098011 | 720 | pSB1T3 | Tetracycline | N/A |
OmpR (R. sphaeroides) | BBa_K227010 | 720 | ? | ? | N/A |
Terminator | BBa_B0015 | 129 | pSB1AK3 | Ampicillin and Kanamycin | plate 1, 23L |
OmpR + Terminator | sequence | 916 | pany-amp | Ampicillin | synthesized |
OmpC promoter | BBa_R0082 | 108 | pSB1A2 | Ampicillin | plate 1, 16K |
puc promoter | BBa_K227007 | 651 | ? | ? | N/A |
puc B/A | sequence | 375 | pSB1A3 | Ampicillin | N/A |
puc B | YP_353390 | 156 | ? | ? | N/A |
puc A | YP_353391 | 165 | ? | ? | N/A |
OmpC promoter+BA | sequence | 539 | pany-kana | Kanamycin | synthesized |
Light Response System | BBa_M30109 | 4,333 | pSB1AC3 | Ampicillin and Chloramphenicol | source plate 1022, 8C |
TetR repressible | BBa_J13002 | 74 | pSB1A2 | Ampicillin | plate 1, 13B |
Green Fluorescent Protein | BBa_E0240 | 876 | pSB1A2 | Ampicillin | plate 1, 12M |
Plasmids used to create and characterize our project
Plasmid | Base Pairs | Resistance | Copy Number |
pSB1A2 | 2,079 | Ampicillin | high |
pSB1K3 | 2,206 | Kanamycin | high |
pSB1A3 | 2,157 | Ampicillin | high |
pSB2K3 | 4,425 | Kanamycin | variable |
pSB1T3 | 2,463 | Tetracycline | high |
pSB1AK3 | 3,189 | Ampicillin and Kanamycin | high |
pANY | |||
pRK404 | |||
pRKPLHT7 | Tetracycline | ||
pRKCBC3 | Tetracycline |
Parts submitted to the Registry of Standard Biological Parts
Component | Part/Accession # | Base Pairs | Plasmid | Resistance |
RBS+Cph8 | BBa_K227000 | 2,256 | plasmid | Resistance |
RBS+Cph8+RBS+OmpR+Terminator | BBa_K227001 | 3,139 | plasmid | Resistance |
Cph8 (resubmission) | BBa_I15010 | 2,238 | plasmid | Resistance |
RBS+Cph8+RBS+OmpR+Terminator +OmpC promoter+pucBA | BBa_K227003 | 3,639 | plasmid | Resistance |
puc A | BBa_K227004 | 165 | plasmid | Resistance |
puc B | BBa_K227005 | 156 | plasmid | Resistance |
puc BA | BBa_K227006 | 376 | plasmid | Resistance |
puc promoter | BBa_K227007 | 651 | plasmid | Resistance |
OmpC promoter + GFP | BBa_K227008 | 992 | plasmid | Resistance |
RBS+Cph8+OmpC+GFP | BBa_K227009 | 3,256 | plasmid | Resistance |
OmpR (optimized for Rhodobacter Sphaeroides) | BBa_K227010 | 720 | plasmid | Resistance |
The first part of our characterization begins with the puc promoter. The puc promoter is what turns on the entire system naturally in Rhodobacter sphaeroides. The puc promoter is what ultimately controls the number of LH2 light harvesting complexes, which is how our system will yield an increase in photosynthetic efficiency. It is important that we are able to compare the transcription rate of the puc promoter in the two systems so that we can determine exactly how much efficiency is gained by adding a red light sensor. By attaching Green Fluorescent Protein (GFP) to the promoter we can quantify the rate of transcription by measuring the emission of green light using a fluorescence spectrophotometer. We would expect to see more fluorescence with more transcription and vis versa.
Modeling the Gene Regulatory Network
References
1. Alon, Uri. Introduction to systems biology and the design principles of biological networks. Boca Raton, FL: Chapman & Hall, 2006.
2. Bower, James M. Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology). New York: M.I.T. PRESS, 2001.
3. System modeling in cellular biology from concepts to nuts and bolts. Cambridge, MA: MIT P, 2006.