Part:BBa_K3102042
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2958
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
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.
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.
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