Difference between revisions of "Part:BBa M50004:Experience"
 
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<I>Stanford BioE44 "Team WERK" 12.10.2016</I>
 
<I>Stanford BioE44 "Team WERK" 12.10.2016</I>
 
<BR>We transformed E. coli with both BBa_M50004 and BBa_M50005 in an attempt to replicate the first step of A. borkumensis' alkane degradation in different bacteria. Under proper induction (IPTG), our Western blot protocol revealed moderate levels of alkB1 production. However, pH testing failed to confirm the cooperative function of alkB1 and alkG, with transformed bacteria in alkane-rich conditions behaving relatively indistinguishably from those in standard conditions. This may have been due to a failure in double transformation, with the two proteins being expressed at disproportionate levels.  
 
<BR>We transformed E. coli with both BBa_M50004 and BBa_M50005 in an attempt to replicate the first step of A. borkumensis' alkane degradation in different bacteria. Under proper induction (IPTG), our Western blot protocol revealed moderate levels of alkB1 production. However, pH testing failed to confirm the cooperative function of alkB1 and alkG, with transformed bacteria in alkane-rich conditions behaving relatively indistinguishably from those in standard conditions. This may have been due to a failure in double transformation, with the two proteins being expressed at disproportionate levels.  
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[[File:FLAG_2.jpg]]
 
[[File:FLAG_2.jpg]]
  
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<BR> This image shows the results of our Western Blot protocol (using an anti-His antibody). Low levels of alkB1 expression from singly-transformed bacteria are visible in the highlighted region; however, doubly-transformed bacteria expressed hardly any alkB1 production.
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<BR> The above image shows the results of our Western Blot protocol (using an anti-FLAG antibody). Low levels of alkB1 expression from singly-transformed bacteria are visible in the highlighted region; however, doubly-transformed bacteria expressed hardly any alkB1 production.
  
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<BR>
 
[[File:Alkane Sum.jpg]]
 
[[File:Alkane Sum.jpg]]
  
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<BR> This graph shows the results of our pH testing; each dataset represents a different instance of transformed E. coli. Measurements were normalized against the initial pH of each bacteria culture. Under proper collaborative function of the alkG and alkB1 enzymes, pH ought to have decreased over time relative to the control; however, no concrete results were observed.  
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<BR> The above graph shows the results of our pH testing; each dataset represents a different instance of transformed E. coli. Measurements were normalized against the initial pH of each bacteria culture. Under proper collaborative function of the alkG and alkB1 enzymes, pH ought to have decreased over time relative to the control; however, no concrete results were observed.  
  
 
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===Stanford Location===
 
===Stanford Location===
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Plasmid name: ABO-2707 <BR>DNA2.0 Gene #: pD444-CF<BR>Organism: E. coli<BR>Device type: Actuator<BR>Glycerol stock barcode: 0133027153<BR>Box label: BioE44 F16
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Plasmid name: ABO_2707 <BR>DNA2.0 Gene #: pD444-CF<BR>Organism: E. coli<BR>Device type: Actuator<BR>Glycerol stock barcode: 0133027153<BR>Box label: BioE44 F16

Latest revision as of 22:33, 11 December 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_M50004

BBa_M50004 contains the alkB1 protein (ABO_2707) originating in the bacterium Alcanivorax borkumensis. It is meant to be used in conjunction with BBa_M50005 (alkG protein, ABO_2708) and other enzymes from A. borkumensis to degrade alkanes measuring up to 32 carbons as part of A. borkumensis' digestive process.

User Reviews

Stanford BioE44 "Team WERK" 12.10.2016
We transformed E. coli with both BBa_M50004 and BBa_M50005 in an attempt to replicate the first step of A. borkumensis' alkane degradation in different bacteria. Under proper induction (IPTG), our Western blot protocol revealed moderate levels of alkB1 production. However, pH testing failed to confirm the cooperative function of alkB1 and alkG, with transformed bacteria in alkane-rich conditions behaving relatively indistinguishably from those in standard conditions. This may have been due to a failure in double transformation, with the two proteins being expressed at disproportionate levels.


FLAG 2.jpg


The above image shows the results of our Western Blot protocol (using an anti-FLAG antibody). Low levels of alkB1 expression from singly-transformed bacteria are visible in the highlighted region; however, doubly-transformed bacteria expressed hardly any alkB1 production.


Alkane Sum.jpg


The above graph shows the results of our pH testing; each dataset represents a different instance of transformed E. coli. Measurements were normalized against the initial pH of each bacteria culture. Under proper collaborative function of the alkG and alkB1 enzymes, pH ought to have decreased over time relative to the control; however, no concrete results were observed.

UNIQ1275f6a832174d09-partinfo-00000000-QINU UNIQ1275f6a832174d09-partinfo-00000001-QINU

Stanford Location

Plasmid name: ABO_2707
DNA2.0 Gene #: pD444-CF
Organism: E. coli
Device type: Actuator
Glycerol stock barcode: 0133027153
Box label: BioE44 F16