Difference between revisions of "Part:BBa K1985004"

(→‎Validation)
(→‎Validation)
 
(18 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1985004 short</partinfo>
 
<partinfo>BBa_K1985004 short</partinfo>
  
−
This part is an differentiation on Part:[https://parts.igem.org/Part:BBa_K1985000 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.
+
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.
  
  
Line 12: Line 12:
 
===Usage and Biology===
 
===Usage and Biology===
  
−
This part has a his-tag included so was used to purify the expressed mamP protein. MamP 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.
+
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.
 
It was expressed, purified and then exposed to iron.
 
For more information on its biology and usage, see part BBa_K1985001.
 
For more information on its biology and usage, see part BBa_K1985001.
Line 20: Line 20:
 
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.
 
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.
  
−
[[File:PSB1C3--SecS-sol-MamT- gel.jpeg||400px|thumb|centre|Figure 2. Agarose gel of the restriction digest of BBa_K1985004 in pSCB13, with EcoRI and PstI.]]
+
[[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.]]
  
  
−
The proteins were then visualised with EM under different conditions: a reducing environment and with magnetite crystals. The samples were concentrated but protein could be seen.
+
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.
  
−
INSERT EM IMAGE
 
  
−
An absorbance spectra was produced that shows peaks at xxx compared to the control.
+
[[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.]]
  
−
INSERT SPECTRA AND CONTROL.
 
  
  
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
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]]
 +
 +
 +
 +
 +
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.
 +
 +
[[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.]]
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

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.