PASantiago Chile/notebook

Notebook

March 2016 to March 2017:

1. We work with BioBricks of previous years (kit iGEM 2015. 2016).
2. We re-suspend genetic material and start to do all the standard methods, like
3. The results are not what expected. We cannot see the material in the gel, doing impossible to prove our biological circuit.

April 2017-July 2017

1.We received the distribution kit 2017.
2.We prove again with all the procedure, but we cannot have the results that expected.

July 11th-13th

1.Review and later shipment of parts to IDT to synthesize (glocks Gene Fragments)
2. The parts were:

  • LuxR + LuxI (Lux)
  • AmilCp + Lemon scent (Module 2)
  • cI repressor + Prm + Or + Pr + Cro (Complete sensor)
  • cI repressor + Prm (Part 1, module 1)
  • Or + Pr + Cro (Part 2, module 1)
  • Saturday 19th, August

    1.Re-suspension of the gBlocks (20ng/ul)
    2.Digestion of parts. (1 hour for 37°C)

  • Complete sensor (SpeI)
  • Lux (XbaI)
  • Plasmid 2 (EcoRI-PstI)
  • Backbone pSB1C3 (EcoRI-PstI)
  • Digestion Protocol: (One enzyme)

    1-H2O Mq                      16,3 ul
    2-Buffer MC                   2    ul 
    3-BSA                             0,2 ul 
    4-DNA                             1   ul 
    5-Enzyme                      0,5 ul
                       Total: 20ul
    

    Digestion Protocol: (Two enzyme)

    1-H2O Mq            15,8 ul
    2-Buffer MC          2    ul  
    3-BSA                     0,2 ul 
    4-DNA                     1   ul 
    5-Enzyme 1           0,5 ul 
    6-Enzyme 2           0,5 ul 
                       Total: 20ul
    

    Digestion Protocol: (Backbone)

    1-H2O Mq              15,8 ul 
    2-Buffer H              2    ul  
    3-BSA                     0,2 ul
    4-DNA                     1   ul
    5-Enzyme PstI       0,5 ul  
    6-Enzyme EcoRI    0,5 ul_ 
                       Total: 20ul
    

    Wednesday 23rd

    1-Ligation. (for 3 hours at 37°C)

  • module 2 with Backbone

  • Ligation Protocol: (gblock + backbone)

    1-H2O Mq            2 ul 
    2-Gblock              4ul 
    3-Backbone         2ul 
    4-Buffer ligase     1ul 
    5-T4 Ligase           1ul​
                       Total: 10ul
    

    Saturday 26th

    1-Ligation of complete sensor + Lux 2-Digestion

  • Part 1 (SpeI)
  • Part 2 (XbaI-SpeI)

  • 3-Transformation of module 2 + backbone (3hours at room temperature)

    ● Transformation protocol (Work in burner)
              * Note: It is important to keep all materials at 4 ° C (on ice) during the procedure. 
              1. In a sterile tube (1.5ml or 2.0ml) add 50μl of calcium-competent bacteria. 
              2. Add 2μl of DNA (BioBrick) and mix with light taps. 
              3. Rest on ice for 20 minutes to 30 minutes. 
              4. Give a heat shock of 42 ° C to the tubes for 1 minute. 
              5. Place on ice for 2 minutes. 
              6. Add 200μl of LB medium and incubate at 37 ° C for 20 minutes to 30 minutes. 
              7. Cultivate in falcon tubes and incubate at 37 ° C 
    

    4-Cultivate transformation of module 2 + backbone

    ● Cultivation protocol (Work in burner) 
              1. Fill n falcon tubes (where "n" is the number of samples to be cultured) and a control 
                  falcon tube with 5ml of LB. 
              2. Leave aside the control tube. 
              3. Pipes containing bacteria will be added with 100μl of bacteria already transformed. 
              4. To tubes containing bacteria add 1μl of antibiotic (Clor or Amp) per 1ml of LB. 
              5. Finally incubate at 37 ° C. 
    

    5-Electrophoresis module 2 + backbone. We do not see the band. Possible problem with sample concentration.

    Saturday 2nd, September

    1- Ligation:
    ● Part 1, module 1 + part 2, module
    ● Complete sensor -lux + backbone
    2- Double digestion, for 3 hours at 37°C, then 15 minutes at 65°C

  • Module 2 (EcoRI-PstI)
  • 3-Results past week: Cultivate of module 2 + backbone, good, clean control.
    4-Electrophoresis:
    We cannot see the bands, again. The problem is dissolution.

    Wednesday 6th

    1-Ligation part1-2, module 2 + lux (let at 37°C for 3 hours)
    2-Electrophoresis to all the samples of IDT without digest, two backbones of different years.

    3-Calcium Competent bacteria are done for use

    Saturday 9th

    1-Ligation (let at 37°C for three hours)

  • Part 1, module 1- part 2, module 1 – lux + backbone
  • Module 2 + backbone

  • 2-Transformation of complete sensor + lux + backbone
    3-Plasmidial extraction of cultivate module 2 + backbone

    ● Plasmidial Extraction protocol 
              1. Add 1000 μl of sample into a sterile tube (1.5ml or 2.0ml). 
              2. We centrifuge samples at maximum speed for 5 minutes. 
              3. Remove the liquid to leave only the pellet (sample). 
              4. Add 250 μl of resuspension solution and resuspend.  
              5. Add 250 μl of lysis solution and invert the sample 4 times. 
              6. We added 10 μl of alkaline protease and inverted 4 times 
              7. Incubate 5 minutes at room temperature 
              8. Add 350 μl of neutralizing solution and inverted 4 times.  
              9. Spin at full speed for 10 minutes. 
              10. We insert 600 μl of the supernatant into a column and it is in a collection tube. 
              11. We centrifuged at full speed for 1 minute. 
              12. Remove lysate and reinsert the column into the collection tube. 
              13. Add 750 μl of washing solution. 
              14. Spin at full speed for 1 minute. 
              15. We discard the lysate and reinsert in the column.  
              16. Add 250 μl of wash solution. 
              17. Spin at full speed for 3 minutes.
              18. Transfer the column to a sterile tube (1.5ml or 2.0ml).
              19. Add 50 μl of nuclease-free water (MQ Water). 
              20. Spin at full speed for 3 minutes. 
              21. Store samples at -20ºC. 
    

    4-Electrophoresis: we see a little bit the bands. The problem is with sample concentrations.

    Thursday 21st

    1-Plasmidial extraction of module 2 + backbone
    2-Electrophoresis

    Friday 22nd

    1-Ligation of part 1, module 1 + part 2, module 1
    2-Digestion of Lux
    3-Plasmidial extraction of module 2 + backbone (x2)
    4-Electrophoresis of plasmid 2 complete and part1 and 2 of module 1

    Saturday 23rd

    1-Ligation

  • Part 1, module 1- part 2, module 1 + lux (New)
  • Part 1, module 1- part 2, module 1 + lux (old)

  • 2-Re- cultivate

  • Module 2 + backbone
  • Complete sensor + lux + backbone

  • 3-Digestion with EcoRI of all the finished plasmids (Plasmidial Extraction)

  • Complete sensor + lux + backbone (09/22)
  • Module 2 + backbone (09/22)

  • Module 2 + backbone (09 /02)

  • Module 2 + backbone (09/02)

  • Wednesday 27th

    1- Ligation of part 1 and 2 of module 1 + lux + backbone
    2- Electrophoresis

  • Complete sensor + lux + backbone, we cannot see it.

  • Module 2 + backbone, we can see the band, it is necessary to verify molecular weight with double digestion (EcoRI- PstI)

  • Saturday 30th

    1-Double digestion with EcoRI and PstI for verify molecular weight of module 2 + backbone.
    2-Transformation of part 1 and part 2 of module 2 + lux. Then cultivate in a new plates and eppendorf tubes
    3-Results of re-cultivate. Clean control and growth uniform
    4-Plasmidial extraction :

  • Re-cultivate module 2 + backbone

  • Complete sensor + lux + backbone.
  • 5-Electrophoresis results:

  • We just see the band with extraction of the complete sensor + lux + backbone and one of the two double digestion.

  • The problem, once again is about concentration
  • Wednesday 4th, October

    1-Plasmidial extraction of transformation part 1 and 2 (module 1) + lux + backbone (three different tubes)
    2-Electrophoresis. We cannot see the bands.

    Saturday 7th

    1-Plasmidial Extraction (Javier) of all the tubes with good growth.
    2-Restriction with EcoRI and PstI.

  • Part 1, Module 1

  • Part 2, module 1
  • Complete sensor
  • Lux
  • Module 2
  • 3-cultive again in a new agar plate :

    ●transformation of part1 +part 2+lux +backbone

    Saturday 14th

    1- Plasmidial extraction

  • Module 2 + backbone

  • Complete sensor + lux + backbone
  • Part 1 and 2 of module 1 + lux + backbone
  • 2-Re-cultivate
    3-Digestion of gblocks of moldule 1.
    4-We detect an error with all the double digestion. Then we start to do all again.

    Wednesday 18th

    1-Digestion

  • Backbone (EcoRI- PstI)

  • Module 2 (EcoRI- PstI)
  • Part 1, module 1 (EcoRI)
  • Part 1, module 1 (EcoRI)
  • Complete sensor (EcoRI)
  • Saturday 21st

    1-Ligation

  • Module 2 + backbone

  • Complete sensor + lux
  • Part 1, module 1 + part 2, module 1
  • Lux + backbone
  • 2-Electrophoresis of all digestions and ligations. We cannot see the bands.

    Wednesday 25th

    1- Change in the protocols of digestion and ligation, for get more concentration of dna and can see bands in electrophoresis.

    double Digestion(new protocol)

    H2O Mq    4,8 ul 
    Buffer        2ul
    Bsa             0,2 ul  
    DNA           11ul 
    enzyme1    1ul ​
    enzyme2     1ul 
                       Total: 20ul
    

    ligation (new protocol)

    dna 1         8ul 
    dna 2         8ul 
    buffer ligase 2ul 
    t4 ligase 3ul 
                       Total: 20ul
    

    2. Digestion:

  • part 1, module 1

  • part 2, module 1
  • lux
  • complete sensor
  • module 2
  • backbone
  • 3. ligation

  • part 1 + part 2

  • lux + backbone
  • Thursday 26th

    1. ligation part 1 + part 2 + lux +backbone
    2. electrophoresis of digested parts of yesterday.

    Results: we can saw the band and then check if the molecular weight was right. (WE CONFIRM THAT CONCENTRATION WAS THE PROBLEM)

    Friday 27th

    Digestion:

  • part 1, module 1

  • part 2, module 1
  • lux
  • complete sensor
  • module 2
  • backbone
  • 2. ligation

  • part 1 + part 2

  • lux + backbone
  • module 2 +backbone
  • complete sensor + lux
  • 3. electrophoresis

  • ligation part 1 + part 2 + lux +backbone

  • double digestion of backbone
  • Results : the ligation was not good, the molecular weight was wrong. Maybe the problem was the quantity of ligase, so we add 2ul of t4 ligase.

    the molecular weight of the backbone was right, so the double digestion and the new protocol works.

    Saturday 28th

    1. ligation

  • part 1 + part 2 + lux + backbone

  • complete sensor + lux + backbone
  • module 2 + backbone
  • 2. electrophoresis

    results:

  • the first band has the right molecular weight. ligation part1+part2 was the correct

  • second band, has less molecular weight (1600bp less) something rare at 6.000bp
  • third band, 1600 bp approx less
  • seventh band, is rare, not good bands
  • ligation with extra t4, has the wrong weight (4th band)
  • all the ligations have 1600 bp less
  • module 2+backbone, we cannot saw the band.
  • Conclusions:

  • the digestion of lux parts was wrong (part molecular weight :1600 bp approx)

  • Just part 1 + part 2 was right.












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