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. 
          1. Leave aside the control tube. 
          1. Pipes containing bacteria will be added with 100μl of bacteria already transformed. 
          1. To tubes containing bacteria add 1μl of antibiotic (Clor or Amp) per 1ml of LB. 
          1. 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.

PASantiago Chile/notebook