Difference between revisions of "Part:BBa K2205004"

Latest revision as of 20:14, 27 October 2017

T7-Sarcosine Oxidase

Sarcosine Oxidase under the control of an IPTG-inducible T7 promoter. Catalyses sarcosine to yield glycine, formaldehyde and hydrogen peroxide.

Induction with IPTG

Sarcosine Oxidase Expression: Sarcosine Oxidase expression was induced by adding 40 µl of 100 mM IPTG. Lane 1: 6 µl ladder, Lane 2: 10 µl sfGFP, Lane 3: BL21-DE3, Lane 4: 10µl SOX 1, Lane 5: 10 µl SOX 2, Lane 6: 10 µl SOX 3, Lane 7: 10 µl SOX 4, Lane 8: 10 µl SOX 5, Lane 9: 10 µl SOX 6, Lane 10: 6 µl ladder.

Formaldehyde Production

Testing of Sarcosine Oxidase with Sarcosine: Lysed cell cultures induced with 40 µl of a 100 mM stock of IPTG tested with formaldehyde strips. From left to right: SOX+0 µl sarcosine, SOX+IPTG+0 µl sarcosine, WT+0 µl sarcosine, WT+IPTG+0 µl sarcosine, SOX+50 µl sarcosine, SOX+IPTG+50 µl sarcosine, WT+50 µl sarcosine, WT+IPTG+50 µl sarcosine, SOX+200 µl sarcosine, SOX+IPTG+200 µl, WT+200 µl sarcosine, WT+IPTG+200 µl.

This shows sarcosine oxidase works as expected, however there is leaky expression as formaldehyde is produced when no IPTG is added.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 Sarcosine Oxidase under the control of an IPTG-inducible T7 promoter. Catalyses sarcosine to yield glycine, formaldehyde and hydrogen peroxide.
+==Induction with IPTG==
+[[File:T--Newcastle--Correct sox protein gel.png|400px|]]
+'''Sarcosine Oxidase Expression:''' Sarcosine Oxidase expression was induced by adding 40 µl of 100 mM IPTG. Lane 1: 6 µl ladder, Lane 2: 10 µl sfGFP, Lane 3: BL21-DE3, Lane 4: 10µl SOX 1, Lane 5: 10 µl SOX 2, Lane 6: 10 µl SOX 3, Lane 7: 10 µl SOX 4, Lane 8: 10 µl SOX 5, Lane 9: 10 µl SOX 6, Lane 10: 6 µl ladder.
+==Formaldehyde Production==
+[[File:T--Newcastle--SOX testing.JPG|400px|]]
+'''Testing of Sarcosine Oxidase with Sarcosine:''' Lysed cell cultures induced with 40 µl of a 100 mM stock of IPTG tested with formaldehyde strips. From left to right: SOX+0 µl sarcosine, SOX+IPTG+0 µl sarcosine, WT+0 µl sarcosine, WT+IPTG+0 µl sarcosine, SOX+50 µl sarcosine, SOX+IPTG+50 µl sarcosine, WT+50 µl sarcosine, WT+IPTG+50 µl sarcosine, SOX+200 µl sarcosine, SOX+IPTG+200 µl, WT+200 µl sarcosine, WT+IPTG+200 µl.
+This shows sarcosine oxidase works as expected, however there is leaky expression as formaldehyde is produced when no IPTG is added.