Difference between revisions of "Part:BBa K2905001"

Latest revision as of 16:02, 18 October 2019

Constitutively expressed 6-His-TEV-MttB

Trimethylamine methyltransferase (MttB), under control of constitutive promoter (J23101) and ribosome binding site (B0034). Contains 6 His tag and TEV protease site.

Measuring Protein Expression: Alma 2019

To analyze protein expression for this part, we grew strains containing the indicated BioBrick overnight in LB + Chloramphenicol. This culture was then diluted in fresh media to an OD600 of 0.2, grown for an additional hour, and pelleted by centrifugation. The pellet was resuspended in B-PER reagent (Thermo-Scientific) containing PMSF, lysozyme and DNase. From this, soluble (S) and in-solube (I) protein fractions were obtained and resolved on a 12% SDS PAGE gel. A representative gel is below.

The expected sizes of the products were MttB: 36.5 kDa without pyrrolysine, MttC: 22.5 kDa, MtbA: 36.5 kdA. Based on these results, we conclude that the expression of our BioBricks is low or non-existent; it is below our ability to detect using this method.

To try and recover the minute amounts of protein that may have been produce, we ran our solube protein lysates (derived either from DH5a, as above, or BL21 DE3) over a Ni-NTA column; this will capture the H6 tagged protein in this BioBrick. A small amount of resin (100uL) was loaded with the lysate. Fractions were collected for flowthrough (F), a 200uL wash (W), and two separate 200uL elutions (E1 and E2), and these were resolved on a 12% SDS PAGE gel, a representative shown below.

Unfortunately, we were unable to recover detectable amounts of H6 tagged protein using this approach. Our expression is either very low or non-existent, and/or the H6 tagged protein is ending up as in-soluble protein.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 104
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 1220
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 869
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1046

@@ Line 5: / Line 5: @@
 Trimethylamine methyltransferase (MttB), under control of constitutive promoter (J23101) and ribosome binding site (B0034). Contains 6 His tag and TEV protease site.
-<!-- Add more about the biology of this part here
+===Measuring Protein Expression: Alma 2019===
-===Usage and Biology===
+To analyze protein expression for this part, we grew strains containing the indicated BioBrick overnight in LB + Chloramphenicol. This culture was then diluted in fresh media to an OD600 of 0.2, grown for an additional hour, and pelleted by centrifugation. The pellet was resuspended in B-PER reagent (Thermo-Scientific) containing PMSF, lysozyme and DNase. From this, soluble (S) and in-solube (I) protein fractions were obtained and resolved on a 12% SDS PAGE gel. A representative gel is below.
+[[File:T--Alma--sds_page1.png|600px]]
+The expected sizes of the products were MttB: 36.5 kDa without pyrrolysine, MttC: 22.5 kDa, MtbA: 36.5 kdA. Based on these results, we conclude that the expression of our BioBricks is low or non-existent; it is below our ability to detect using this method.
+To try and recover the minute amounts of protein that may have been produce, we ran our solube protein lysates (derived either from DH5a, as above, or BL21 DE3) over a Ni-NTA column; this will capture the H6 tagged protein in this BioBrick. A small amount of resin (100uL) was loaded with the lysate. Fractions were collected for flowthrough (F), a 200uL wash (W), and two separate 200uL elutions (E1 and E2), and these were resolved on a 12% SDS PAGE gel, a representative shown below.
+[[File:T--Alma--sds_page2.png|600px]]
+Unfortunately, we were unable to recover detectable amounts of H6 tagged protein using this approach. Our expression is either very low or non-existent, and/or the H6 tagged protein is ending up as in-soluble protein.
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