Difference between revisions of "Part:BBa K4187022"
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4187022 parameters</partinfo>
 
<partinfo>BBa_K4187022 parameters</partinfo>
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[[File:File.jpg]]
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We assessed the ability of E.Coli BL21 transformed with the amyH (amyH BL21) gene to degrade starch by performing a lugol staining assay on solid cultures. As this amylase’s activity depends on NaCl concentrations, we tested multiple salt concentrations (i.e. 0%, 5%). We cultured the bacteria as well-defined separated colonies on solid media containing starch. We defined degradation halos as the total unstained surface minus the surface of the colony: S(unstained) - S(colony).
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We measured these surfaces using ImageJ, an open source software developed by Wayne Rasband.
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[[File:Characterization AmyH 0%.jpeg]]
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Figure 1: Starch concentration: 0.2 g/L NaCl concentration: 0% (w/v)  NT BL21 mean halo surface = 0.7 cm²; amyH BL21 mean halo surface = 1.28 cm²
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[[File:Characterization AmyH.jpeg]]
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Figure 2: Starch concentration: 0.2 g/L NaCl concentration: 5% (w/v)  NT BL21 mean halo surface = 0.05 cm²; AmyH BL21 mean halo surface = 1.25 cm²
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Our results show an increased starch degradation activity in E.coli BL21 transformed with AmyH as the mean halo sizes of the colonies are higher in transformed colonies.
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We cannot conclude on the NaCl concentration effect on the amylase activity as the maximum mean halo sizes are similar in both NaCl concentration conditions. However we observe that with a 5% NaCl concentration, halo sizes of non-transformed BL21 are close to zero while halo sizes of transformed BL21 reach the same value as, in the 0% NaCl condition.
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Figure 1: Picture of Lugol staining assay comparing wt BL21 to amyH BL21. Starch concentration: 0.2 g/L NaCl concentration: 0% (w/v)  NT BL21 mean halo surface = 0.7 cm²; amyH BL21 mean halo surface = 1.28 cm²
 
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Revision as of 11:44, 12 October 2022


T7-RBS-AMYH-T1-T7TE


AmyH is an alpha-amylase from the bacterium Halomonas meridiana that cuts the 1,4-linkage of two molecules of glucose. In our system, it allows e.coli to efficiently use starch as a source of glucose.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 259
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 205