Difference between revisions of "Part:BBa J33201"

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1. Growth curve of E.coli containing part K577881 in different concentrations of arsenite(As3+)  
 
1. Growth curve of E.coli containing part K577881 in different concentrations of arsenite(As3+)  
 
Constructed plasmid pSB1C3-pArsR-GFP containing J33201 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of arsenite(As3+) solutions were added into the test tubes, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm and 100ppm). Samples were taken at different time points of 0h, 1h, 2h…8h and overnight (16h) at one hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader.  
 
Constructed plasmid pSB1C3-pArsR-GFP containing J33201 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of arsenite(As3+) solutions were added into the test tubes, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm and 100ppm). Samples were taken at different time points of 0h, 1h, 2h…8h and overnight (16h) at one hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader.  
 +
 
2. GFP expression of E.coli under the control of J33201 induced by different concentration of arsenite (As3+)
 
2. GFP expression of E.coli under the control of J33201 induced by different concentration of arsenite (As3+)
 
Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0,5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm).  
 
Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0,5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm).  
 
(1) Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time. The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager.
 
(1) Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time. The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager.
 
(2)Samples were taken at different time points of 0h, 2h, 4h, 6h and overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.
 
(2)Samples were taken at different time points of 0h, 2h, 4h, 6h and overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.
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<h2> Results<h2>
 
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Fig.1 showed that bacteria growth was not affected in the range of 5ppb-10ppm of arsenite solution (As3+). However, as the concentration of arsenite increased to 50ppm, the growth of the bacteria was seriously inhibited, suggesting the toxictity to cells at this level.
 

Revision as of 08:30, 19 October 2019


E. coli chromosomal ars promoter with arsR repressor gene

This part consists of the promoter of the E. coli JM109 chromosomal arsenic detoxification operon (ars operon), including the ArsR repressor binding site and the arsR gene encoding the arsR repressor protein, together with its ribosome binding site. Addition of any other genes to the 3' end of this part will result in their expression being dependent on the presence of sodium arsenate or sodium arsenite. Arsenite or arsenite anion binds to the repressor protein ArsR, resulting in inability to repress the promoter. Based on our experiments, a concentration of 1 micromolar sodium arsenate in LB is sufficient for essentially full expression, though this will vary according to conditions.

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 255
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Supplementary material offered by other groups

The following is from TJU-China 2018. We used the part:Bba-J33201 saved in plate3 and wanted to build a new arsenic induction loop through it. Therefore, we first added 10μlddH2O to the corresponding hole, and then took PCR was performed at 1μl, and the corresponding DNA fragment was obtained. 

  T--TJU_China--1-8.png

Figure1. The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker. Lane 1-6，Bba-J33201

Then we performed PCR on the promoter fragment and ArsR fragment in this fragment, and the results are as follows. 

  T--TJU_China--1-9.png

Figure2. The result of nucleic acid gel electrophoresis of Ars Promoter after PCR. lane M, Marker. Lane 1-4, Ars Promoter. 

  T--TJU_China--1-10.png

Figure 3. The result of nucleic acid gel electrophoresis of ArsR Protein after PCR. Lane M, Marker. Line1, ArsR Protein.

Characterization

Worldshaper-Wuhan 2019's Characterization


Aim of experiment

Based on J33201 part, a biosensor pSB1C3-pArsR-RBS-GFP(K3153000) was constructed to detect arsenite(As3+) in water and contributed to this well-characterized part. 1. Growth curve of E.coli containing part K577881 in different concentrations of arsenite(As3+) Constructed plasmid pSB1C3-pArsR-GFP containing J33201 part was transformed into E.coli DH5α strain. Single colony was selected to inoculate LB broth containing chloramphenicol and cultured overnight. Then overnight culture was inoculated in the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of arsenite(As3+) solutions were added into the test tubes, respectively, so that the final concentration of arsenic was (0, 5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm and 100ppm). Samples were taken at different time points of 0h, 1h, 2h…8h and overnight (16h) at one hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader. 2. GFP expression of E.coli under the control of J33201 induced by different concentration of arsenite (As3+) Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of arsenite solutions were added into the test tube, respectively, so that the final concentration of arsenic was (0,5ppb, 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, 10ppm, 50ppm, 100ppm). (1) Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time. The bacteria were centrifuged, and the pellets were was observed and photographed under Blue Light Gel Imager. (2)Samples were taken at different time points of 0h, 2h, 4h, 6h and overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.