Difference between revisions of "Part:BBa K4252001"
 
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==Introduction==
 
==Introduction==
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So far, only one type of P exporting protein, YjbB, has been reported in E. coli.The mechanism of phosphorus export by yjbB is still unclear. But based on experimental phenomena we choose YjbB as the phosphorus output element in our project.
+
So far, only one type of P exporting protein, YjbB, has been reported in E. coli [1]. The mechanism of phosphorus export by yjbB is still unclear. But based on experimental phenomena we choose YjbB as the phosphorus output element in our project.
  
 
==Engineering==
 
==Engineering==
−
DNA fragments containing yjbB was amplified from E. coli MG1655 genomic DNA using
+
DNA fragments containing yjbB was amplified from E. coli MG1655 genomic DNA using the primers yjbB-5’/yjbB-3.The PCR fragments were inserted into the Xbal/Xhol sites of the vector pET-22b(+). And the vector was transformed into BL21.
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the primers yjbB-5’/yjbB-3’.The PCR fragments were inserted into the Xbal/Xhol sites of pET-22b(+).
+
 
 +
==Design==
 +
Functional validation method
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The yjbB-BL21 strain was amplified to about OD600=0.7 in LB medium containing 1 / 1000 ampicyl resistance, induced protein expression with 2mM IPTG for 2 hours, The bacteria were washed twice with phosphorus-free MOPs medium, and continued induction with 2mM IPTG to measure the supernatant phosphorus concentration over time (1-7 hours) and OD value (0.1 per increase).
  
 
==Result==
 
==Result==
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===Results of pET-22b(+)-yjbB sequencing (primers: YjbB-2-5′, YjbB-2-3′)
 
 
<br>
 
<br>
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[[File: results-of-pet-22b-yjbB-sequencing.png|500px|thumb|center|Figure 1: results-of-pet-22b-yjbB-sequencing.]]
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[[File: The phosphorus concentration in the supernatant changed with time.png|500px|thumb|center|Figure 1: The change of phosphate concentration in the supernatant with the increase of time. When BL21-yjbB and wild-type BL21 grow in LB culture medium until OD600 is about 0.7, IPTG was added until the final concentration was 2mM and induced for 2 hours. The bacteria were resuspended in 50ml phosphate-free medium, and 2mM IPTG was added to continue induction. The turbidity of bacteria in the experimental group hardly changed.]]
 +
 
 +
As shown in the figure above, the concentration of wild type group phosphate is basically maintained at a stable value. From the fifth hour, the phosphate concentration in the culture of the experimental group was much higher than that of the wild type
 +
group, and increased significantly with time. It is proved that the phosphorus export element is feasible.
 +
 
 +
==References==
 +
*[1]Kei Motomura, Ryuichi Hirota, Nobuteru Ohnaka, Mai Okada, Takeshi Ikeda, Tomohiro Morohoshi, Hisao Ohtake, Akio Kuroda, Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation, FEMS Microbiology Letters, Volume 320, Issue 1, July 2011, Pages 25–32, https://doi.org/10.1111/j.1574-6968.2011.02285.x

Latest revision as of 11:00, 11 October 2022


phosphate exportation

As a phosphorus export element.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 426
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1174


Introduction

So far, only one type of P exporting protein, YjbB, has been reported in E. coli [1]. The mechanism of phosphorus export by yjbB is still unclear. But based on experimental phenomena we choose YjbB as the phosphorus output element in our project.

Engineering

DNA fragments containing yjbB was amplified from E. coli MG1655 genomic DNA using the primers yjbB-5’/yjbB-3.The PCR fragments were inserted into the Xbal/Xhol sites of the vector pET-22b(+). And the vector was transformed into BL21.

Design

Functional validation method The yjbB-BL21 strain was amplified to about OD600=0.7 in LB medium containing 1 / 1000 ampicyl resistance, induced protein expression with 2mM IPTG for 2 hours, The bacteria were washed twice with phosphorus-free MOPs medium, and continued induction with 2mM IPTG to measure the supernatant phosphorus concentration over time (1-7 hours) and OD value (0.1 per increase).

Result


Figure 1: The change of phosphate concentration in the supernatant with the increase of time. When BL21-yjbB and wild-type BL21 grow in LB culture medium until OD600 is about 0.7, IPTG was added until the final concentration was 2mM and induced for 2 hours. The bacteria were resuspended in 50ml phosphate-free medium, and 2mM IPTG was added to continue induction. The turbidity of bacteria in the experimental group hardly changed.

As shown in the figure above, the concentration of wild type group phosphate is basically maintained at a stable value. From the fifth hour, the phosphate concentration in the culture of the experimental group was much higher than that of the wild type group, and increased significantly with time. It is proved that the phosphorus export element is feasible.

References

  • [1]Kei Motomura, Ryuichi Hirota, Nobuteru Ohnaka, Mai Okada, Takeshi Ikeda, Tomohiro Morohoshi, Hisao Ohtake, Akio Kuroda, Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation, FEMS Microbiology Letters, Volume 320, Issue 1, July 2011, Pages 25–32, https://doi.org/10.1111/j.1574-6968.2011.02285.x