Difference between revisions of "Part:BBa K1951011"
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To test the correct functioning of this biobrick we have performed three different types of experiment. | To test the correct functioning of this biobrick we have performed three different types of experiment. | ||
Revision as of 20:30, 19 October 2016
The purpose of this biobrick is to produce desferrioxamine B siderophore by a controled manner, for controlled potential toxicity.
This biobrick was made from 6 parts:
Available in the registry :
Designed by our team :
Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known.
Contents
A precious metal binder
Previous work showed that siderophores are able to catch other metals by default. Especifically, many articles showed a high affinity of Desferrioxamine B ( produce by Streptomyces coelicolor) for tetravalent metal ions like platinum. These results encouraged us to make a biobrick coding the sequence corresponding to the 4 enzymes involved on the metabolic pathway of Desferrioxamine B. E. coli was used to produce this biobrick. Produce a gram positive bacteria pathway in a gram negative bacteria is restrictive [1] , considering the risk of toxicity for the conductress bacteria. To counter this potential issue, we regulate trasnscription using the control of an inductible promotor (pBAD/araC).
A medical treatment
Desferrioxamine B is a really well referenced siderophore.
Deferoxamine acts by binding free iron in the bloodstream and enhancing its elimination in the urine. By removing excess iron, the agent reduces the damage done to various organs and tissues, such as the liver. Also, it speeds healing of nerve damage (and minimizes the extent of recent nerve trauma).
- It is currently used of on the World Health Organization's List of Essential Medicines, the most important medications needed in a basic health system. But it is used in the treatment of many others diseases as well.
- Desferrioxamine B may modulate expression and release of inflammatory mediators by specific cell types.
Design sum up
To ensure the good protein quality and solubility :
- Coding sequence we have designed previouslyBBa_K1951000BBa_K1951001BBa_K1951002BBa_K1951003 have been optimized for E. coli
- The promotor and RBS we have used I0500 and B0034 were designed and tested in previous studies and demonstrate as inductible. Inducted, protomer allows a medium level expression in E.coli
SLIC oligo table
| DesA slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGCGCTCGCACTTGCTT |
| desA slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGAGGCACGGTC |
| desB slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGGTATTGGTCTTGGG |
| desB slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTACACTGCAAATTC |
| desC slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCTCGCCTTTCCACG |
| desC slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGCTGAAACCGC |
| desD slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGAGTTTAGCTGATGCA |
| des D slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACGCCCGGCTAA |
Experience sum up
To test the correct functioning of this biobrick we have performed three different types of experiment.
- Proteins production checked by SDS PAGE
- Mesure of cadaverine production tested by HPLC
Proteins production
Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway as illustrated here.
HPLC test
We made a big composite part able to produce every proteins involved in the biosynthetic pathway of the desferrioxamine B- ↑ Wandersman & Delepaire https://www.ncbi.nlm.nih.gov/pubmed/15487950