Difference between revisions of "Part:BBa K562009"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K562009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K562009 SequenceAndFeatures</partinfo>
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===Results===
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<i>E. coli</i> was transformed with <partinfo>BBa_K562009</partinfo> and <partinfo>BBa_K562013</partinfo> and grown anaerobically for 16 hours in LB medium containing 0.4% (w/v) glucose. Cells were harvested, washed and broken in 50 mls B-PER solution before being loaded onto an IMAC column. Bound proteins were eluted with an imidazole gradient and analysed by SDS-PAGE.
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Fractions contained all the components of the synthetic microcompartment. The bands shown were positively identified by tryptic peptide mass fingerprinting. In addition, the co-expressed GFP was also identified around 26 kDa. Note that the banding pattern changed depending on whether the sample was boiled. In unboiled samples the PduB protein (which is produced as full-lenth PduB and truncated PduB') remains in an oligomeric state and appears larger than expected on the gel.
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[[Image:BBa_K562009_Prot_Data.jpg]]
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:Figure 1: Isolation of a synthetic bacterial microcompartment by metal affinity chromatography. Each of PduB, PduU, PduN, and PduK carry hexa-histidine tags. PduA, PduT,a nd PduJ are not tagged. GFP was co-expressed as a PduD40-GFP-HA fusion and could be detected in the sample. This means that the initial 40 amino acids of Pdu40 can target GFP into the microcompartment.
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Revision as of 16:34, 21 September 2011

Salty_PduABTUNJK BMC Components

This is a composite part comprising a constitutive promoter, which is the tatABCD promoter from E. coli K-12, driving production of the PduA, PduB, PduT, PduU and PduN proteins from Salmonella enterica serovar Typhimurium LT2 (identical to part BBa_K562008) AND proteins PduJ and PduK. These are all components of the Salmonella bacterial microcompartment (BMC). The PduB, PduU, PduN and PduK gene products all carry C-terminal 6-His affinity/epitope tags. Production of the PduB, PduU, PduN and PduK proteins has been verified by Western immunoblotting (anti-penta-His) and production of PduA, PduB, PduT, PduU, PduN, PduJ and PduK has been confirmed by 35S-Met labelling. The proteins have also been purified from E. coli by immobilised metal affinity chromatography (IMAC) and identified by tryptic peptide mass fingerprinting. The construct is cloned as an EcoRI / PstI fragment into pSB1C3. The clone is also known as pSB-ABTUNJK in the Sargent Laboratory, Dundee, UK.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1898
    Illegal BamHI site found at 3058
    Illegal BamHI site found at 3197
    Illegal BamHI site found at 3291
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1441
    Illegal NgoMIV site found at 2685
    Illegal AgeI site found at 1426
    Illegal AgeI site found at 2040
    Illegal AgeI site found at 2952
    Illegal AgeI site found at 2988
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

E. coli was transformed with BBa_K562009 and BBa_K562013 and grown anaerobically for 16 hours in LB medium containing 0.4% (w/v) glucose. Cells were harvested, washed and broken in 50 mls B-PER solution before being loaded onto an IMAC column. Bound proteins were eluted with an imidazole gradient and analysed by SDS-PAGE.

Fractions contained all the components of the synthetic microcompartment. The bands shown were positively identified by tryptic peptide mass fingerprinting. In addition, the co-expressed GFP was also identified around 26 kDa. Note that the banding pattern changed depending on whether the sample was boiled. In unboiled samples the PduB protein (which is produced as full-lenth PduB and truncated PduB') remains in an oligomeric state and appears larger than expected on the gel.

BBa K562009 Prot Data.jpg

Figure 1: Isolation of a synthetic bacterial microcompartment by metal affinity chromatography. Each of PduB, PduU, PduN, and PduK carry hexa-histidine tags. PduA, PduT,a nd PduJ are not tagged. GFP was co-expressed as a PduD40-GFP-HA fusion and could be detected in the sample. This means that the initial 40 amino acids of Pdu40 can target GFP into the microcompartment.