Difference between revisions of "Part:BBa K1985004"

 
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<partinfo>BBa_K1985004 short</partinfo>
 
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Usage: The mamT gene produces a protein that can be used to transfer electrons to iron molecules. It can be used in combination with other mam genes to form an electron transport complex (reference) that should form magnetic magnetite crystals when the protein is exposed to into in solution. This sequence produces a soluble protein because it has the membrane anchor cleaved and is targeted to the secretory pathway, which will leave the protein in the periplasm. It was expressed, purified and then exposed to iron.(??)
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This part is an differentiation on Part:[https://parts.igem.org/Part:BBa_K1985001 BBa_K1985001]. The wild type sequence was altered to remove the membrane anchor so that the part is soluble and a SecS sequence was added to target the protein to the periplasm. A his-tag was added for easier protein purification.
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Biology:
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MamT is a proposed cytochrome protein (reference) due to the presence of a heme binding motif within its sequence. It forms a complex with other Mam proteins on the membrane of the native species, Magnetospirillum gryphiswaldense. Together they promote magnetite crystal maturation within an organelle called the magnetosome.  
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K1985004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1985004 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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This part has a his-tag included so was used to purify the expressed mamT protein. MamT is usually targeted to the membrane, however in this part the targeting sequence has been cleaved and it should instead be targeted to the periplasm.
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It was expressed, purified and then exposed to iron.
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For more information on its biology and usage, see part BBa_K1985001.
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===Validation===
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The part was first validated with a diagnostic restriction digest using EcoRI and PstI and agarose gel electrophoresis. The expected band sizes from the digest were: 2029 base pairs for the plasmid backbone and 604 for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.
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[[File:PSB1C3--SecS-sol-MamT- gel.jpeg||400px|thumb|centre|Figure 1. Agarose gel of the restriction digest of BBa_K1985004 in pSB1C3, with EcoRI and PstI.]]
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SDS-PAGE images revealed bands in all gels that were not present in the before induction samples and the control. For MamT the band indicated by the arrow equates to the, 17 kDa, soluble MamT protein with his-tag.
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[[File:T--Kent--mamTsds1.jpg||400px|thumb|left|Figure 2. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.]] [[File:T--Kent--mamTsds2.jpg||400px|thumb|right|Figure 3. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.]]
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The magnetite particles were imaged under TEM with solutions of mamT along with mamP and mamX under anaerobic conditions. [[File:DaniieTEMnewpic.png||400px|thumb|centre|Figure 4. EM image of magnetite crystals with hypothesised new crystals nucleated circled in red, these crystals are in the presence of the three proteins mamT, P and X]]
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The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of all three proteins as well as the control. MamT has a distinct increase in absorbance to the control at 280nm displaying an increase in protein.
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[[File:Allprotein graph.jpeg||400px|thumb|centre|Figure 5. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX, P and T as well as the control. For mamT (Blue) a distinct peak was seen compared to the control (grey) at 280nm.]]
  
 
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Latest revision as of 17:43, 27 October 2016

mamT his-tagged, signal sequence cleaved

This part is an differentiation on Part:BBa_K1985001. The wild type sequence was altered to remove the membrane anchor so that the part is soluble and a SecS sequence was added to target the protein to the periplasm. A his-tag was added for easier protein purification.


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 NgoMIV site found at 138
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This part has a his-tag included so was used to purify the expressed mamT protein. MamT is usually targeted to the membrane, however in this part the targeting sequence has been cleaved and it should instead be targeted to the periplasm. It was expressed, purified and then exposed to iron. For more information on its biology and usage, see part BBa_K1985001.

Validation

The part was first validated with a diagnostic restriction digest using EcoRI and PstI and agarose gel electrophoresis. The expected band sizes from the digest were: 2029 base pairs for the plasmid backbone and 604 for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.

Figure 1. Agarose gel of the restriction digest of BBa_K1985004 in pSB1C3, with EcoRI and PstI.


SDS-PAGE images revealed bands in all gels that were not present in the before induction samples and the control. For MamT the band indicated by the arrow equates to the, 17 kDa, soluble MamT protein with his-tag.


Figure 2. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.
Figure 3. Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.



















The magnetite particles were imaged under TEM with solutions of mamT along with mamP and mamX under anaerobic conditions.
Figure 4. EM image of magnetite crystals with hypothesised new crystals nucleated circled in red, these crystals are in the presence of the three proteins mamT, P and X



The his-tagged proteins were purified by nickle affinity chromatography. An absorbance spectra was produced of all three proteins as well as the control. MamT has a distinct increase in absorbance to the control at 280nm displaying an increase in protein.

Figure 5. UV spectra of nickle affinity chromatography protein fractions from E.coli expressing mamX, P and T as well as the control. For mamT (Blue) a distinct peak was seen compared to the control (grey) at 280nm.