Difference between revisions of "Part:BBa K4614002"
 
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<partinfo>BBa_K4614002 short</partinfo>
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4614002 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4614002 parameters</partinfo>
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  We used T7 promoter to induce IPN and R5(part BBa_K461400) expression, IPN, a carrier protein from the genome of Pseudomonas syringae, used to display R5 on the surface of bacteria.
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  We used T7 promoter to induce Lnak and R5(part BBa_K461400) expression. IPN,a protein encoded by Lnak，a carrier protein from the genome of Pseudomonas syringae,was used to display R5 on the surface of bacteria.
 
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  In use, the modified E. coli can be cultured to the logarithmic stage, and IPTG with a final concentration of 1.0 ug/mL can be added to induce 3 h, and protein expression can be completed
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  In use, the modified <i>E. coli</i> can be cultured to the logarithmic stage, and IPTG with a final concentration of 1.0 µg/mL can be added to induce 3 h, and protein expression can be completed
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We constructed an expression vector to express R5 and its surface display carrier protein IPN fusion protein, using T7 promoter as the promoter, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37 °C at a final concentration of 1.0 µg/mL in the logarithmic phase<sup>[1]</sup>, disrupted the bacteria, and performed Western blotting experiments on the supernatant and precipitation of the cell disruption solution to verify the expression of the protein of interest.
 
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    <img src="https://static.igem.wiki/teams/4614/wiki/si-parts/wb.png" width="500" height="auto" class="centered-image">
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<p class="figurelegend">Fig1.Bacterial holoprotein Western blotting development result</p>
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We silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group did the same, we collected the silicified bacteria and observed the bacteria using transmission electron microscopy to obtain the silicification effect of R5 under the silicification conditions we used.
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        <img src="https://static.igem.wiki/teams/4614/wiki/si-parts/dianjing.png" width="600" height="auto" class="centered-image">
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<p class="figurelegend">Fig2.Transmission electron microscope image
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According to transmission electron microscopy observations, we found that R5 anchored on the surface of <i>E. coli</i>, catalyzed the hydrolysis of TEOS into silica in the TEOS silicification system, and deposited on the bacterial surface, forming a silica shell on the R5-<i>E. coli</i> surface, while changing the permeability of the cell membrane, allowing TEOS to enter the cell and generate silicon-filled cells. The experimental group bacteria cells had normal morphology, were filled with silicon, and had intact and smooth cell walls. The control group bacteria cells had less intracellular material, incomplete cell walls, and varying degrees of deformation. To some extent, this indicates that bacterial silicification can maintain the cell shape and provide a certain rigidity.
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===Usage and Biology===
 
  
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===References of CAU_China===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4614002 SequenceAndFeatures</partinfo>
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[1]薛双红. 基于细菌表面展示技术的功能性无机材料合成研究[D].武汉理工大学,2019.
  
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===Functional Parameters===
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===Usage and Biology===
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<partinfo>BBa_K4614002 parameters</partinfo>
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Latest revision as of 15:25, 12 October 2023


Lnak-R5

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 529
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 529
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 529
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 529
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
    Illegal AgeI site found at 679
  • 1000
    COMPATIBLE WITH RFC[1000]


We used T7 promoter to induce Lnak and R5(part BBa_K461400) expression. IPN,a protein encoded by Lnak，a carrier protein from the genome of Pseudomonas syringae,was used to display R5 on the surface of bacteria.

In use, the modified E. coli can be cultured to the logarithmic stage, and IPTG with a final concentration of 1.0 µg/mL can be added to induce 3 h, and protein expression can be completed

We constructed an expression vector to express R5 and its surface display carrier protein IPN fusion protein, using T7 promoter as the promoter, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37 °C at a final concentration of 1.0 µg/mL in the logarithmic phase[1], disrupted the bacteria, and performed Western blotting experiments on the supernatant and precipitation of the cell disruption solution to verify the expression of the protein of interest.

Fig1.Bacterial holoprotein Western blotting development result

We silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group did the same, we collected the silicified bacteria and observed the bacteria using transmission electron microscopy to obtain the silicification effect of R5 under the silicification conditions we used.

Fig2.Transmission electron microscope image

According to transmission electron microscopy observations, we found that R5 anchored on the surface of E. coli, catalyzed the hydrolysis of TEOS into silica in the TEOS silicification system, and deposited on the bacterial surface, forming a silica shell on the R5-E. coli surface, while changing the permeability of the cell membrane, allowing TEOS to enter the cell and generate silicon-filled cells. The experimental group bacteria cells had normal morphology, were filled with silicon, and had intact and smooth cell walls. The control group bacteria cells had less intracellular material, incomplete cell walls, and varying degrees of deformation. To some extent, this indicates that bacterial silicification can maintain the cell shape and provide a certain rigidity.

References of CAU_China

[1]薛双红. 基于细菌表面展示技术的功能性无机材料合成研究[D].武汉理工大学,2019.