Difference between revisions of "Part:BBa K3102042"
 
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<partinfo>BBa_K3102042 short</partinfo>
 
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This cellulose acetate degradation (ACE, EG1, TEV, HlyA) composite part is  composed of acetate esterase (CAE):BBa_K3102023, endo-1,4-B-glucanase I (EG1):BBa_K3102024, and TEV protease (TEV):BBa_K3102025. This part allows the secretion of these enzymes directly in the extracellular medium using E. coli type I secretion system.
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This "cellulose acetate degradation" composite part refered as CET is  composed of acetate esterase (cae):BBa_K3102023, endo-1,4-B-glucanase I (eg1):BBa_K3102024, and TEV protease (tev):BBa_K3102025. This part allows the secretion of these enzymes directly in the extracellular medium using E. coli type I secretion system.
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We used an inducible promoter T7 BBa_I719005 in order to control CAE, EG1 and Tev production thanks to IPTG induction.  
  
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We used an inducible promoter T7 BBa_I719005 in order to control CAE, EG1 and TEV production thanks to IPTG induction.
 
  
 
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<partinfo>BBa_K3102042 parameters</partinfo>
 
<partinfo>BBa_K3102042 parameters</partinfo>
 
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<h1>Degradation of cellulose acetate with engineered bacteria</h1>
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<p>A critical step of our system is the degradation of cellulose acetate by our modified <em>Escherichia coli</em>. From this enzymatic process, glucose and acetate are produced and used as energetic substrates by <em>E. coli </em>itself. The degradation is based on the secretion of Cellulose Acetate Esterase (<em>cae</em>) and Endo-1 4- β-glucanase I (<em>eg1</em>). This biobrick will be refered as CET.</p>
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<p>Thus, to prove that degradation is functional, pH and acetate concentrations are measured. Experiment’s details can be found in “Protocol: Acetate Detection”.</p>
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<h2>Measurement of pH</h2>
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<p>If cellulose acetate degradation is functional, then acetate accumulation is occurring. Thus, pH acidification should be detected. pH indicator strips are used to measure pH overtime. Below are presented the first and last measures.</p>
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<p><img src="https://static.igem.org/mediawiki/parts/4/44/T--Ionis_Paris--CET_image1.png" alt="pH evolution chart" />
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<em>Figure 1: pH evolution (1. t=0h; 2. t=24h; a. pH scale for 1; b. pH scale for 2.)</em></p>
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<p>Those data show an alteration of pH for all three samples: regarding the control a slight increase is observed, certainly due to regular metabolism. However, concerning EG1 and CET, pH is becoming more acidic overtime. This would indicate a functional degradation of cellulose acetate fibers. CET seems to be more efficient as pH is lower.</p>
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<h2>Measurement of acetate concentration</h2>
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<p>To ensure that the diminution of pH is due to an accumulation of acetate, we use an Acetate Detection Kit. We tested the kit on EG1, CET transformed bacteria, and non-transformed <em>E. coli </em>bacteria.</p>
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<p><img src="https://static.igem.org/mediawiki/parts/3/34/T--Ionis_Paris--CET_2.jpg" alt="Bacteria comparison chart" />
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<em>Figure 2: Comparison of the bacteria performance in degrading cellulose acetate</em></p>
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<p>Those data show that the enzymatic process is functional. For both EG1 and CET transformed bacteria, acetate is detected and measured whereas the control does not seem to have any degradation activity. This complements the pH measurement data described above.</p>
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<p>Those results demonstrate the increased efficiency of our BioBrick over the previous one, EG1, as the acetate concentration secreted by CET modified bacteria is higher.</p>
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Latest revision as of 13:53, 21 October 2019


Cellulose Acetate degradation (CAE, EG1, Tev, HlyA)

This "cellulose acetate degradation" composite part refered as CET is composed of acetate esterase (cae):BBa_K3102023, endo-1,4-B-glucanase I (eg1):BBa_K3102024, and TEV protease (tev):BBa_K3102025. This part allows the secretion of these enzymes directly in the extracellular medium using E. coli type I secretion system.

We used an inducible promoter T7 BBa_I719005 in order to control CAE, EG1 and Tev production thanks to IPTG induction.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2958
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 124
    Illegal NgoMIV site found at 1234
    Illegal NgoMIV site found at 4120
    Illegal NgoMIV site found at 4426
    Illegal AgeI site found at 254
    Illegal AgeI site found at 341
    Illegal AgeI site found at 479
    Illegal AgeI site found at 662
    Illegal AgeI site found at 710
    Illegal AgeI site found at 878
    Illegal AgeI site found at 1091
  • 1000
    COMPATIBLE WITH RFC[1000]


Degradation of cellulose acetate with engineered bacteria

A critical step of our system is the degradation of cellulose acetate by our modified Escherichia coli. From this enzymatic process, glucose and acetate are produced and used as energetic substrates by E. coli itself. The degradation is based on the secretion of Cellulose Acetate Esterase (cae) and Endo-1 4- β-glucanase I (eg1). This biobrick will be refered as CET.

Thus, to prove that degradation is functional, pH and acetate concentrations are measured. Experiment’s details can be found in “Protocol: Acetate Detection”.

Measurement of pH

If cellulose acetate degradation is functional, then acetate accumulation is occurring. Thus, pH acidification should be detected. pH indicator strips are used to measure pH overtime. Below are presented the first and last measures.

pH evolution chart Figure 1: pH evolution (1. t=0h; 2. t=24h; a. pH scale for 1; b. pH scale for 2.)

Those data show an alteration of pH for all three samples: regarding the control a slight increase is observed, certainly due to regular metabolism. However, concerning EG1 and CET, pH is becoming more acidic overtime. This would indicate a functional degradation of cellulose acetate fibers. CET seems to be more efficient as pH is lower.

Measurement of acetate concentration

To ensure that the diminution of pH is due to an accumulation of acetate, we use an Acetate Detection Kit. We tested the kit on EG1, CET transformed bacteria, and non-transformed E. coli bacteria.

Bacteria comparison chart Figure 2: Comparison of the bacteria performance in degrading cellulose acetate

Those data show that the enzymatic process is functional. For both EG1 and CET transformed bacteria, acetate is detected and measured whereas the control does not seem to have any degradation activity. This complements the pH measurement data described above.

Those results demonstrate the increased efficiency of our BioBrick over the previous one, EG1, as the acetate concentration secreted by CET modified bacteria is higher.