BME494 Project Group9

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Contents

ABSTRACT

Vitamin D
Vitamin D



Vitamin D is essential for our health. In addition to our diets, humans also create vitamin D from the sun. Our proposed design will potentially have the ability to determine the presence of 25(OH) D3 in blood. This could potentially evolve into a diagnostic device which can be an effective substitute for the current testing methods of Vitamin D levels.

















BACKGROUND

Vitamin D is found in fish, eggs, fortified milk and cod liver oil.
Vitamin D is found in fish, eggs, fortified milk and cod liver oil.



Sunlight contributes to Vitamin D production because the human skin can synthesize Vitamin D3 when exposed to UVB rays from the Sun. Vitamin D is essential for maintaining normal blood levels of both calcium and phosphorous. The absorption of calcium due to Vitamin D strengthens bones to prevent diseases that cause skeletal deformities such as Rickets and muscular/skeletal degradation such as Osteomalacia.


The diseases that Vitamin D deficiency cause affect children and older people mostly. The lack of Vitamin D leads to other complications.


The proposed design has the potential to be used as a simple and efficient diagnostic test.





PROOF OF CONCEPT DESIGN

In 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.


  • New Natural Part: The homo sapien kidney mitochondria enzyme 25 Hydroxyvitamin D3 1-alpha-hydroxylase (CYP27B1) is being used in this application for its co-transcription factors and promoter. The promoter and co-transcription factors are activated specifically by 25(OH)D3 and the parathyroid hormone (PTH), both which have the ability to be present in the blood.
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


  • 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.



Assembly Scheme

  • Step One: In the diagram below, it is clear that natural parts from the homo sapien CYP27B1 are cut via EcoRI and SpeI while the desried GFP is cut with the same restriction enzymes. At the same time, the RBS specific for the GFP is cut via EcoRI and PstI along with the terminator being cut in the same way.
  • 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.


Image:Plasmid.JPG



Illustration of assembly.



  • 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).


Illustration of D-Vitameter.






TESTING


Measurement


In order to test the functionality of the proposed system, two different concentrations of 25(OH)D3 will be added with the transformed E. Coli solution. One concentration is 60 ng/ml of 25(OH)D3, which describes the optimal, healthy amount for an individual. This data will be compared with a concentration of 40 ng/ml, which describes the deficient, or at risk, amount for an individual.


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.



Expected Observations

Image:Absorbancy.JPG



Results from spectrophotometer illustrating specific peaks for GFP.


Image:group9graph2.jpg

When 25(OH)D3 is present (60 ng/ml), ideally there would be a 100% GFP concentration.



Tuning Our System


There are a few areas in which our system can be modified:


For one, the RBS for GFP we are using is already biobricked by a prior iGEM team. By adding different RBS's with different strengths, the efficacy of the system should increase/decrease in GFP expression accordingly; such that with a weaker GFP RBS, the GFP read in the spectrophotometer will be less. With a stronger GFP RBS, the spectrophotometer will read a greater GFP absorbancy.


Secondly, this system only is using five of the most relevant co-transcription factors form the CYP27B1 enzyme when in fact there are actually over 200! It is predicted that the addition of more co-transcription factors will increase the GFP expression, and thus increase the GFP absorbancy.


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.




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.


•The meter will be user friendly and structured similarly to our current glucose meters.


•Normal Levels of Vitamin D may slightly vary with each individual; the meter is not more than a medical tool to indicate deficiency, and it cannot replace a doctor.


•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

Carolina Tostado
Carolina Tostado
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










Robbia Hendrix
Robbia Hendrix
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!







Alex Medawar
Alex Medawar
Junior in Biomedical Engineering, SBHSE.





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