BME494 Project Group9: Difference between revisions
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<div class=noprint style="width: 900px; height: 369px; color: #565051; background-color: #dcdcdc">[[Image: | <div class=noprint style="width: 900px; height: 369px; color: #565051; background-color: #dcdcdc">[[Image:Vitd.JPG]]</div> | ||
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==PROOF OF CONCEPT DESIGN== | ==PROOF OF CONCEPT DESIGN== | ||
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Prior to primer creation, it was determined if there was the need for site directed mutagenesis. Fortunately, through the aid of Nebcutter, it was determined that the original gene set was ok to use as is. | Prior to primer creation, it was determined if there was the need for site directed mutagenesis. Fortunately, through the aid of Nebcutter, it was determined that the original gene set was ok to use as is. | ||
Natural Part Primer: | |||
Forward: 5'-ctttctttcgcgagcaccctct-3' | |||
Reverse: 5'-acagtttttggacagatagtcc-3' | |||
BioBricked Primer: | |||
Forward: 5'-gaattcgcggccgcttcttctctagagctttctttcgcgagcacc-3' | |||
Reverse: 5'-ctgcagcgccggcgtactagtaacagtttttggacagatagtcc-3' | |||
No site directed mutagenesis necessary | |||
GC content 40%, within range | |||
* '''Existing BioBricks''':The pre-existing Biobrick part BBa_101015 is used and includes a RBS, GFP, and a terminator. The 2008 Minnesota iGem team has biobricked this part and found it successful in their projects, thus it is expected to work correctly in our system. | * '''Existing BioBricks''':The pre-existing Biobrick part BBa_101015 is used and includes a RBS, GFP, and a terminator. The 2008 Minnesota iGem team has biobricked this part and found it successful in their projects, thus it is expected to work correctly in our system. | ||
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* '''Step Two''': The transformed natural part and RBS plasmid is cut with EcoRI and SpeI, while the transformed GFP and terminator plasmid is cut with XbaI and PstI. A destination plasmid is used and cut via EcoRI and PstI to allow for the insertion of two transformed plasmid pieces. | * '''Step Two''': The transformed natural part and RBS plasmid is cut with EcoRI and SpeI, while the transformed GFP and terminator plasmid is cut with XbaI and PstI. A destination plasmid is used and cut via EcoRI and PstI to allow for the insertion of two transformed plasmid pieces. | ||
Ampicillin and Kanamycin are utilized to help select for the appropriate plasmids durring testing. | * Ampicillin and Kanamycin are utilized to help select for the appropriate plasmids durring testing. | ||
[[Image:Plasmid.JPG]] | [[Image:Plasmid.JPG]] | ||
Illustration of assembly. | |||
* '''Future Vision''':D-Vitameter | * '''Future Vision''': D-Vitameter | ||
Ideally this would be a device that would benefit from the proposed technology. This compact and portable device would allow patients to get a reading of their ability to produce Vitamin D by just using one drop of blood. The basic components of this small device would be an enclosed box with a small polycarbonate window. The device will allow to introduce a sample in a microscope slide and shine a UV light to detect presence of 25(OH). | Ideally this would be a device that would benefit from the proposed technology. This compact and portable device would allow patients to get a reading of their ability to produce Vitamin D by just using one drop of blood. The basic components of this small device would be an enclosed box with a small polycarbonate window. The device will allow to introduce a sample in a microscope slide and shine a UV light to detect presence of 25(OH). | ||
[[Image:Dvitameter.jpg|600px]] | [[Image:Dvitameter.jpg|600px]] | ||
Illustration of D-Vitameter. | Illustration of D-Vitameter. | ||
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[[Image:Absorbancy.JPG]] | [[Image:Absorbancy.JPG]] | ||
Results from spectrophotometer illustrating specific peaks for GFP. | |||
[[Image:group9graph2.jpg]] | [[Image:group9graph2.jpg]] | ||
When 25(OH)D3 is present, ideally there would be a 100% GFP concentration | When 25(OH)D3 is present (60 ng/ml), ideally there would be a 100% GFP concentration. | ||
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Lastly, we should continue to search for way a to create a repressor that is inhibited when in presence of active Vitamin D3, 1,25(OH). If this is found, a repressor system could be implemented on the existing structure to create another approach to an in home Vitamin D3 diagnostic. | Lastly, we should continue to search for way a to create a repressor that is inhibited when in presence of active Vitamin D3, 1,25(OH). If this is found, a repressor system could be implemented on the existing structure to create another approach to an in home Vitamin D3 diagnostic. | ||
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==HUMAN PRACTICES== | ==HUMAN PRACTICES== | ||
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•This meter will be targetting children and elders in particular because thos age groups are most prone to deficiency. For kids, pricking fingers for small | •This meter will be targetting children and elders in particular because thos age groups are most prone to deficiency. For kids, pricking fingers for small blood samples won't be ideal if it's a routine | ||
==OUR TEAM== | ==OUR TEAM== |
Latest revision as of 20:37, 16 March 2012
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ABSTRACT
BACKGROUND
PROOF OF CONCEPT DESIGNIn the presence of 25(OH)D3 in blood, the co-transcription factor and promoter for the CYP27B1 are activated. When the promoter is activated, it expresses the enzyme that causes a chemical change to the active form of Vitamin D. This active form, 1-25-DiHydroxyVitamin D3, is created in the kidney. If the promoter is activated, then it will express the enzyme that chemically changes to the active form of vitamin D in the kidney.
The genes for the CYP27B1 co-transcription factors and promoter were provided via GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Prior to primer creation, it was determined if there was the need for site directed mutagenesis. Fortunately, through the aid of Nebcutter, it was determined that the original gene set was ok to use as is. Natural Part Primer: Forward: 5'-ctttctttcgcgagcaccctct-3' Reverse: 5'-acagtttttggacagatagtcc-3' BioBricked Primer: Forward: 5'-gaattcgcggccgcttcttctctagagctttctttcgcgagcacc-3' Reverse: 5'-ctgcagcgccggcgtactagtaacagtttttggacagatagtcc-3' No site directed mutagenesis necessary GC content 40%, within range
Assembly Scheme
Ideally this would be a device that would benefit from the proposed technology. This compact and portable device would allow patients to get a reading of their ability to produce Vitamin D by just using one drop of blood. The basic components of this small device would be an enclosed box with a small polycarbonate window. The device will allow to introduce a sample in a microscope slide and shine a UV light to detect presence of 25(OH). Illustration of D-Vitameter.
TESTING
Both samples will be run through a spectrophotometer to measure absorbency at wavelength peaks specific to GFP. A max peak of GFP absorbancy is expected around 395nm in addition to a minor absorbancy at 475nm.
When 25(OH)D3 is present (60 ng/ml), ideally there would be a 100% GFP concentration.
HUMAN PRACTICES•The meter is practical because the blood sample is relatively small and can be extracted at any time. Normal tests for Vitamin D deficiency require laboratory procedures and time.
OUR TEAM
Senior Biomedical Engineering, SBHSE, I am interested in learning the general concepts of synthetic biology and its applications, I will be graduating this semester! http://openwetware.org/wiki/User:Carolina_Tostado
Senior in Biochemistry & Dance. I am excited to learn the role of biochemistry within the field of synthetic biology. I am also graduating this semester!
Junior in Biomedical Engineering, SBHSE.
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