Difference between revisions of "Team:WashU StLouis/Proof"

Line 18: Line 18:
  
 
<p>
 
<p>
−
After identifying the genes and constructing the electron donor overproducing plasmids, proper assays were necessary in order to see if our new cells produced usable forms of electron donors. Not only was it shown that the products were overproduced, but also significant data supported the claim that these products were usable in protein synthesis.  
+
After identifying our genes of interest and constructing the electron donor overproducing plasmids, we performed an assay in order to see if our new cells produced usable forms of reduced electron donors. Our assay showed that not only were the products were overproduced, but also significant data supported the claim that these electron donors were usable in protein synthesis.  
 
</p>
 
</p>
  
 
<p>
 
<p>
−
After analyzing the results of our absorbance data of electron donors, it was decided to go a step further to ensure that these construct produced usable electron donors. A biotin assay was found that offered an indirect way of measuring the amount of reduced electron donors present. Biotin, commonly know as vitamin B7, is produced by a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin[1] (see diagram below).  
+
After analyzing the results of our absorbance data of electron donors, we decided to go a step further to ensure that these constructs produced usable electron donors. Biotin, commonly know as vitamin B7, is produced by a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin[1] (see diagram below). We used a readily available biotin assay to indirectly measure the amount of reduced electron donors present in our cells.
 
</p>
 
</p>
  
Line 28: Line 28:
  
 
<p>
 
<p>
−
By measuring the amount of biotin in the new cells, and recognizing the 1:1 stoichiometric relationship between reduced electron donors and biotin molecules[2], the amount of electron donors present and used can be indirectly measured. Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below.
+
By measuring the amount of biotin in the new cells, and recognizing the 1:1 stoichiometric relationship between reduced electron donors and biotin molecules[2], the relative amount of electron donors used can be determined. Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below.
 
<ul>
 
<ul>
 
<li>When our constructs were in DH10B cells, the <i>fldA</i>, <i>fldA</i>-<i>pfo</i>, <i>petF</i>, and <i>petF</i>-<i>pfo</i> strains produced more biotin than the control DH10B strain at some induction level</li>
 
<li>When our constructs were in DH10B cells, the <i>fldA</i>, <i>fldA</i>-<i>pfo</i>, <i>petF</i>, and <i>petF</i>-<i>pfo</i> strains produced more biotin than the control DH10B strain at some induction level</li>

Revision as of 22:14, 18 October 2016

MENU ▤

Proof of Concept

Measuring biotin production to calculate reduced electron donors

After identifying our genes of interest and constructing the electron donor overproducing plasmids, we performed an assay in order to see if our new cells produced usable forms of reduced electron donors. Our assay showed that not only were the products were overproduced, but also significant data supported the claim that these electron donors were usable in protein synthesis.

After analyzing the results of our absorbance data of electron donors, we decided to go a step further to ensure that these constructs produced usable electron donors. Biotin, commonly know as vitamin B7, is produced by a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin[1] (see diagram below). We used a readily available biotin assay to indirectly measure the amount of reduced electron donors present in our cells.

By measuring the amount of biotin in the new cells, and recognizing the 1:1 stoichiometric relationship between reduced electron donors and biotin molecules[2], the relative amount of electron donors used can be determined. Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below.

  • When our constructs were in DH10B cells, the fldA, fldA-pfo, petF, and petF-pfo strains produced more biotin than the control DH10B strain at some induction level
  • When our constructs were in the ΔaceE knockout cells, the fldA, fldA-pfo, and petF strains all produced more biotin than the control DH10B strain, control ΔaceE knockout strain, and DH10B+constructs strain (petF-pfo data is lacking due to its inability to grow)

Go to the conclusions page to see a complete summary of our results and proof of concept experiments.

References

  1. Lin, Steven, and John E. Cronan. "Closing in on complete pathways of biotin biosynthesis." Molecular BioSystems 7.6 (2011): 1811-1821.
  2. Picciocchi, Antoine, Roland Douce, and Claude Alban. "Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis." Plant physiology 127.3 (2001): 1224-1233.