Difference between revisions of "Part:BBa K5136226"
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====Agarose gel electrophoresis (AGE)==== | ====Agarose gel electrophoresis (AGE)==== | ||
The composite part (BBa_K5136226) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> BL21 (DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (4464 bp) can be observed around 5000bp (Fig. 1). | The composite part (BBa_K5136226) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> BL21 (DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (4464 bp) can be observed around 5000bp (Fig. 1). | ||
| + | <center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/226colony.png" width="400px"></html></center> | ||
| + | <center><b>Fig. 1 Colony PCR of BBa_K5136226_pSB1C3 in <I>E. coli</I> BL21(DE3)</b></center> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 17:08, 30 September 2024
I0500-B0034-inpnc-linker-mt3-linker-mt2a-B0015
Biology
INPNC
Ice nucleoprotein (INP), an outer membrane protein from Pseudomonas syringae, has been used as a surface anchor in many researches. The truncated version of INP, namely INPNC which contains only the N- and C-terminal portion of INP, has excellent capacity to anchor target proteins on the cell membrane (1).
MT3 and MT2A
The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular protein and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn2+, N2+, Pb2+, Hg2+, Cd2+, as well as As3+, but their effectiveness in treating Cr2O72- is not satisfactory. MT2a and MT3 are metallothioneins(MTs) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr2O72-. And MT3 has a better adsorption effect on ordinary metal ions. (2).
Usage and Biology
After deinking, heavy metal ions in the ink will be released into the wastewater. Thus, this wastewater needs harmless treatmentremove before discharging. Hence, we use MTs to treat wastewater to remove heavy metals. And, in order to increase the contact area of MTsand metal ions to improve the adsorption efficiency, we introduce INPNC for surface display of MTs. Thus, this composite part BBa_K5136226 was constructed to express the fuesed protein INPNC-linker-MT3-linker-MT2A which anchored on the surface of bacteria.
Characterization
Agarose gel electrophoresis (AGE)
The composite part (BBa_K5136226) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli BL21 (DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (4464 bp) can be observed around 5000bp (Fig. 1).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NotI site found at 1717 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal BamHI site found at 1566
Illegal BamHI site found at 2211
Illegal BamHI site found at 2466 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1308
Illegal NgoMIV site found at 1641
Illegal AgeI site found at 979
Illegal AgeI site found at 1665 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961