Team:Evry/Notebook/Sensing/proposition

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IGEM Evry 2014

Notebook - Sensing

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Sensor construction bphR2/PbphR1:

  1. Culturing of one colony bphR1 promoter (BBa_K1155001), received from Paris-Saclay's team, in 5ml LB + 5µL Chloramphenicol (Cam)
  2. Incubation at 37°C overnight, 200rpm

Jul 27
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Sensor construction bphR2/PbphR1:

  1. Plate 20µL from the culture of 27th july on LB-agar-Cam (25 mL LB-Agar + 25µL Chloramphenicol)
  2. Incubation of plate at 37°C overnight
  3. A glycerol stock was made with 750ml of culture and 750ml of glycerol 50% and stored at -80°C

Jul 28
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Survivability in molecules which are going to be sensed:
Ranges of concentrations of molecules that we want to sense were made as described in following tables:
pouet
1. Serial dilutions of compound were made

  • Lead
    text to print if image not found
  • Cadmium
    text to print if image not found
  • Nitrite
    text to print if image not found

    2. 300µL of E.coli BL21 was added in each eppendorf tube.
    We had 2 control tubes:
    *A negative control: 2,7mL of MB medium + 300µL of compound
    *A positive control: 2,7mL of MB medium + 300µL of E.coli BL21

    3. Incubation of tubes at 30°C overnight, 200rpm

    Jul 29
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    Survivability in molecules which are going to be sensed - Results:
    Here are the results of the survivability of Pseudovibrio in media with molecules which are going to be sensed:
    pouet
    Pseudovibrio seems to survive in presence of all concentrations of nitrite, it's affect cells' growth but don't kill Pseudovibrio.
    In Lead, big concentrations lead to cells' death, but Pseudovibrio survive very well until a concentration of 5µL.
    Cadmium does'nt seem to block cells' growth in concentrations which we want to use.

    Jul 29

    Picture

    Aug 12
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    Survival test on E.coli BL21:
    10mg of 2 hydroxy,3'-4'dichlorobiphenyl have been received. The compound was lyophilized so it's has been dissolved in 17mL of DMSO in order to have a final concentration of 10(-2)M
    The compound was stored at -20°C, in the dark because the DMSO is sensitive to the light.

    Aug 13
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    Survival test on E.coli BL21:

    This test allowed to know if E.coli can survive at different concentrations of 2 hydroxy-3',4'dichlorobiphenyl

    1. For that, BL21 culture was removed from the -80°C and culturing in 4 mL of LB
    2. E.coli was incubated overnight at 37°C, 200rpm

    Aug 14
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    Survival test on E.coli BL21:

    1. Serial dilution of compound was made from 10(-2) mol/L to 10(-8) mol/L
      image not found
    2. 300µL of E.coli BL21 were added in each eppendorf tube.
      We had 2 control tubes:
      • A negative control which contains 2,7mL of M9 medium + 300µL of compound
      • A positive control which contains 2,7mL of M9 medium + 300µL of E.coli BL21

    3. Tubes were incubated at 37°C overnight, 200rpm

      Aug 15
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    Cell culture of E.coli received from registry
    BBa_K1031211 (Pr-DmpR )
    BBa_K1031221 (P0-RBS B0031-GFP)
    BBa_k1031222 (P0-RBS B0032-GFP)
    Liquid : 3ml of Luria Broth 1x + 34µg/L Chloramphenicol in a 15ml falcon at 37°C shaking
    On plate : 25ml LB 1x + 25ml Agarose 1x + 34µg/L Chloramphenicol
    Observation: DmpR bacteria have difficulties to grow compared with GFP bacteria

    Left : BBa_K1031221 (P0-RBS B0031-GFP)
    Right : BBa_k1031222 (P0-RBS B0032-GFP)

    Aug 15
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    Survival test on E.coli BL21:

    After the incubation of 15th July, bacteria had grown:

    image not found

    Aug 16
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    Aug 17
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    Sensor construction bphR2/PbphR1:

    bphR2 gene, synthetized by Eurofins in a plasmid, was dissolved in 23,5µL of elution buffer because it was lyophilized.
    Concentration obtained = 200ng/µL

    DH5a chimiocompetents were transformed with this plasmid according to this protocol:

    1. Remove DH5a from -80°C (about 200µL) and let them on the ice
    2. Add 100ng of plasmid at DH5a and let during 30mn on ice
    3. Make a heat shock by putting bacteria at 42°C during 30s-1mn
    4. Let 1h at 37°C
    5. Plate 100µL of the culture on LB-agar-Amp
    6. Incubate at 37°C overnight, 200rpm

    Aug 18
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    Dna extraction
    Machery Nagel DNA purification Kit (PROTOCOL)
    PCR using VF2 and VR primer
    Q5 polymerase
    Expected bands :
    DmpR: 2038 bp
    GFP B0031: 1331 bp
    GFP B0032: 1330 bp
    Digestion
    DmpR: SpeI&PstI
    GFP B0031/32: XbaI&PstI


    Analysis
    VF2/VR PCR products were in agreement with the expected size either for DmpR and GFP B0031/32.
    GFP 31/32 digestion products were in agreement with the expected size.
    However DmpR digestion revealed an unexpected profile.

    Gel extraction
    Extraction of GFP B0031/32 digestion product [XbaI-PstI]
    After confirmation of relevant electrophoresis bands, cut the gel all around the band and as close as possible to it.
    Place the resulting piece into a 2ml microcentrifuge tube. Add 1µl of Binding buffer to 1 µg of gel. Place the tube at 55°C to 60°C during 10min or until gel turn completely liquid.
    Follow classical DNA purification protocol.
    Machery Nagel kit (PROTOCOL 2)

    Aug 18
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    Sensor construction bphR2/PbphR1:
    A colony after the DH5a transformation was cultured in 3mL of LB + 3µL Amp, at 37°C, 200rpm, overnight

    Survival test on E.coli BL21:
    Bacteria continue to grow for all concentrations of 2 hydroxy-3',4'dichlorobiphenyl.

    Aug 19
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    PCR using VF2 and VR primer
    Q5 polymerase
    Expected bands :
    DmpR: 2038 bp
    GFP B0031: 1331 bp
    GFP B0032: 1330 bp
    Digestion
    From newly extracted DNA
    DmpR: SpeI&PstI
    GFP B0031/32: XbaI&PstI



    Analysis
    VF2/VR PCR products are still in agreement with the expected size either for DmpR and GFP B0031/32.
    GFP 31/32 digestion products were in agreement with the expected size.
    DmpR digestion by SP is still displaying 2 close bands profile.

    Aug 20
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    Sensor construction bphR2/PbphR1
    We received sequencing reads: that match perfectly to the registry sequence.
    26DJ54
    AATAGGCGTTATCACGAGGCAGAANTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGATGTCCCTGG GTGACATGCGTGATTTGGCCGCCACGCGGATCGCGCTCGAGAGCGAAGCGTTACGCCAAAGCGTGCTGAATGGTGACGCTGAATGGGAGGCGCGGATCGTCAGTTC GTTTCACCGACTGTCATTGATTGAAGAGCCCACGATGCGGGATCCGGCTCGCTGGTTTAATGAGTGGGAGCCAGTCAACCGCGGTTTTCACGAAGCTCTTATCTCT GCCTGTTCGTCCGTCTGGATCCGGCGGTTCCTGTCCATCCTGTATGTGCATATGGAGCGCTACCGCCGATTGACTGCTTACTAGTAGCGGCCGCTGCAGTCCGGCA AAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC GGCTGCGGCGAGCGGTATCA GCTCACTCAGGG

    26DJ55
    CGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGC AGCGGCCGCTACTAGTAAGCAGTCAATCGGCGGTAGCGCTCCATATGCACATACAGGATGGACAGGAACCGCCGGATCCAGACGGACGAACAGGCAGAGATAAGAG CTTCGTGAAAACCGCGGTTGACTGGCTCCCACTCATTAAACCAGCGAGCCGGATCCCGCATCGTGGGCTCTTCAATCAATGACAGTCGGTGAAACGAACTGACGAT CCGCGCCTCCCATTCAGCGTCACCATTCAGCACGCTTTGGCGTAACGCTTCGCTCTCGAGCGCGATCCGCGTGGCGGCCAAATCACGCATGTCACCCAGGGACATC TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA TGATAATAATGGTTTCTTAGA


    Plasmid with bphR2 gene was purified with NucleoSpin Plamsid protocol (Macherey-Nagel) => we obtained 227,5ng/µL
    This plasmid was digested according this protocol:

    1. Add:
      • sterilized water: qsp 20µL
      • template DNA : 500ng
      • buffer 2.1: 2µL
      • BSA: 0,2µL
      • EcoRI: 1µL
      • PstI: 1µL
    2. Reverse for mix
    3. Incubate at 37°C during 45mn
    4. Incubate at 80°C during 20mn
    5. Ligation was not possible because pSB1C3 plasmid was not digested so the digested plasmid (bphR2 gene) was stored at -20°C

    Survival test on E.coli BL21:
    Bacteria survive again for different concentrations but they were very concentrated

    A dilution of the medium has been done for the positive control and the six different concentrations:
    image not found

    Aug 20
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    Sensor construction bphR2/PbpR1
    BBa_J23114 was resuspended with 10 µL sterile water in order to obtain a DNA concentration around 0.2 ng/µl (according to the registry). Solution was transferred into one 1 ml eppendorf tube and stored at -20°C.
    Transformations of constitutive promoter BBa_J23114, RFP BBa_E1010 and terminator BBa_B0015 and vector pSB1C3G3 was done on DH5alpha as followed:

    1. Remove E. coli competent tubes from -80°C and keep it on ice
    2. Add 3 µl of template (here solubilized plasmids from the registry distribution kit) and mix gently
    3. Incubate 10 minutes on ice
    4. Perform an heat shock 30 seconds at 42°C
    5. Incubate 2 minutes on ice
    6. Add 2 ml of LB medium and incubate 60 minutes at 37°C with an agitation at 200 rpm
    7. Plate 200 µl of pSB1C3G3, BBa_B0015 and E1010 on a chloramphenicol LB agar plate and ampicilline LB agar plate for BBa_J23114
    8. Incubate plate overnight at 37°C

    We received primers.
    IMAGE
    Table 4: Received primers with numbers and sequences for PCB sensing constructions



    pSB1C3 was digested with EcoRI and pstI and a ligation with bphR2 was done before the transformation in DH5a.
    50 µL were plated on Cam Lb plate and incubated at 37°C overnight

    Aug 21
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    Transformation plate observation:
    - BBa_E1010: 50-60 colonies
    - BBa_J23114: 150-200 colonies
    - BBa_B0015: 40-50 colonies
    - PSB1C3G3: > 1000 red colonies

    A PCR was performed on 8 colonies for BBa_J23115 (K823012) following the protocol Table 3 and 4.
    Samples are loaded on a 1% agarose gel, 10 µL of sample + 2 µl of loading dye 6X per well. Gel ran 45 minutes at 100 mV.


    Sensor construction bphR2/PbphR1 :
    One colony of the transformation of pSB1C3 was incubated in 3ml LB + 3µL Cam, at 37°C, overnight

    Aug 22
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    Sensor construction bphR2/PbphR1 :

    Miniprep of pSB1C3 was done => 68,9ng/µl and the plasmid was digested with EcoRI-HF and pstI.
    Digestion products of pSB1C3 and bphr2 has been migrated on gel 1X agarosis. Bands expected were:


    • for pSB1C3: one band at 2069bp (vector) and one band at 1070bp (insert)
    • for bphr2: one band at 946 pb (insert) and one band at 1300bp (vector)


    We obtained the good bands so an extraction on gel was done to recover only pSB1C3 without its insert and only bphr2 gene.

    Aug 23
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    Aug 24