Difference between revisions of "Part:BBa K1976053"
 
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         <h1>Colicin E2 DNase trypsin fragment and immunity protein Im2 generator with anderson promoters</h1>
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         <h1>Colicin E2 DNase Trypsin Fragment and Immunity Protein Im2 Generator</h1>
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This parts contains the sequences encoding for the colicin E2 DNase trypsin fragment <a href="https://parts.igem.org/Part:BBa_K197604">BBa_K1976049</a> which can be obtained by trypsin digestion of colicin E2 and the immunity protein Im2 <a href="https://parts.igem.org/Part:BBa_K1976026">BBa_K1976026</a> both under the controle of anderson promoters.
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This part can be used to produce colicin without killing the host cell, as the immunity protein is produced more than equimolar to it.
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Safety is an important aspect in the synthetic biology on one hand to prevent the  transfer of recombinant DNA to natural organisms but on the other hand it is necessary for companies to enable copy protection of the  DNA of their GMO. This part units these points using a new safety mechanism. The DNase fragment of colicin E2 wich enables DNA degradation is inhibited by its immunity protein Im2 wich can only be synthesized under the presence of a non-natural amino acid (nnAA). Cells transformed with this part need a constant addition of the nnAA to survive. If they leave the laboratory environment, the immunity protein will not be synthesized due to the lack of the nnAA and the DNase will degradate the DNA so the genomic information will be lost.
  
 
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===<h2>Characteristics</h2>===
 
  
 
===<h2>Characteristics</h2>===
 
===<h2>Characteristics</h2>===
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The DNase trypsin fragment of colicin E2 consists of 137 residues what conforms to about one third to colicin. We used only one of the three domains of colicin since the metabolic burdon would be significant smaller. This fragment differs to the part 50 in 5 additional amino acids since a trypsin digestion described in the literature leaded to this fragment. So we wanted to check out any difference in the activities between the DNase fragment with and without theese additional amino acids. The activity tests showed that the so called miniColicin has functional DNase activity. The immunity protein has a molecular weight of 10.0 kDa and the modeling of the binding to miniColicin predicted a very strong binding.
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In figure 2 you can see our results of the expression of the DNasedomain. 
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                    Molecular Weight
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<img src="https://static.igem.org/mediawiki/2016/7/7f/T--TU_Darmstadt--Mico_PAGE.png" width="30%">
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                    Colicin E2 Dnase domain
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<b>Figure 2</b>: SDS-PAGE of the Colicin E2 DNase domain, the blue arrow marks the band of the expression. From left to right: Top10 E. coli, Top10 E. coli transformed with BBa_K1976052 and Top10 E. coli transformed with BBa_K1976053.
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                    <br>15.2 kDa<br>
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                    Immunity Protein
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                    <br>10.0 kDa<br>
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<img src="https://static.igem.org/mediawiki/2016/e/e9/T--TU_Darmstadt--miniColi_test.png" width="30%">
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                    Residues
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<b>Figure 3</b>: 1% agarose gel electrophoresis, stained with HD-green.
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The protein product from this part was cleaned up and used in different concentrations to test its activity.
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                    Colicin E2 Dnase domain
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From left to right: pSB1C3 alone, pSB1C3 in the lysate buffer 1x, pSB1C3 in the lysate buffer 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 3x.
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                    133
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                    Colicin E2 Dnase domain
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                    <br>9.06<br>
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                    Immunity Protein
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                    <br>4.66<br>
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                    [Data taken created in Snapgene and http://isoelectric.ovh.org/calculate.php]
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Latest revision as of 03:24, 28 October 2016

Colicin E2 DNase Trypsin Fragment and Immunity Protein Im2 Generator

This parts contains the sequences encoding for the colicin E2 DNase trypsin fragment BBa_K1976049 which can be obtained by trypsin digestion of colicin E2 and the immunity protein Im2 BBa_K1976026 both under the controle of anderson promoters.

Figure 1: The structure of the Im2 immunity protein and Colicin E2 in a complex.


Usage

Safety is an important aspect in the synthetic biology on one hand to prevent the transfer of recombinant DNA to natural organisms but on the other hand it is necessary for companies to enable copy protection of the DNA of their GMO. This part units these points using a new safety mechanism. The DNase fragment of colicin E2 wich enables DNA degradation is inhibited by its immunity protein Im2 wich can only be synthesized under the presence of a non-natural amino acid (nnAA). Cells transformed with this part need a constant addition of the nnAA to survive. If they leave the laboratory environment, the immunity protein will not be synthesized due to the lack of the nnAA and the DNase will degradate the DNA so the genomic information will be lost.

Characteristics

The DNase trypsin fragment of colicin E2 consists of 137 residues what conforms to about one third to colicin. We used only one of the three domains of colicin since the metabolic burdon would be significant smaller. This fragment differs to the part 50 in 5 additional amino acids since a trypsin digestion described in the literature leaded to this fragment. So we wanted to check out any difference in the activities between the DNase fragment with and without theese additional amino acids. The activity tests showed that the so called miniColicin has functional DNase activity. The immunity protein has a molecular weight of 10.0 kDa and the modeling of the binding to miniColicin predicted a very strong binding. In figure 2 you can see our results of the expression of the DNasedomain.

Figure 2: SDS-PAGE of the Colicin E2 DNase domain, the blue arrow marks the band of the expression. From left to right: Top10 E. coli, Top10 E. coli transformed with BBa_K1976052 and Top10 E. coli transformed with BBa_K1976053.


Figure 3: 1% agarose gel electrophoresis, stained with HD-green. The protein product from this part was cleaned up and used in different concentrations to test its activity. From left to right: pSB1C3 alone, pSB1C3 in the lysate buffer 1x, pSB1C3 in the lysate buffer 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 3x.

Characteristics

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 492
    Illegal NheI site found at 515
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 696
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 107
  • 1000
    COMPATIBLE WITH RFC[1000]


[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007