Difference between revisions of "Part:BBa K4187022"

Revision as of 11:47, 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.

File:File.jpg 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).

We measured these surfaces using ImageJ, an open source software developed by Wayne Rasband. 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 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 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. 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. 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 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

@@ Line 6: / Line 6: @@
 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.
+[[File:File.jpg]]
+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).
+We measured these surfaces using ImageJ, an open source software developed by Wayne Rasband.
+[[File:Characterization AmyH 0%.jpeg]]
+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
+[[File:Characterization AmyH.jpeg]]
+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
+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.
+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.
+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
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
@@ Line 17: / Line 28: @@
 ===Functional Parameters===
 <partinfo>BBa_K4187022 parameters</partinfo>
-[[File:File.jpg]]
-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).
-We measured these surfaces using ImageJ, an open source software developed by Wayne Rasband.
-[[File:Characterization AmyH 0%.jpeg]]
-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
-[[File:Characterization AmyH.jpeg]]
-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
-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.
-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.
-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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