Team:Saint Rose School B/Safety


Safety

The team Bio-lignin places great emphasis on safety of synthetic biology. The instructors have particularly introduced importance of biological safety for us, they are going to help us a lot in safety stuff.

Safety in the lab:
Into the lab we have to follow a couple of rules to our safety, since clean our work area until scientific processes, like:

  • You must to take your hair if id too long.
  • Do not smoke, eat or drink in the laboratory.
  • Try not to walk back and forth without reason and, above all, do not run in the laboratory.
  • Always keep your hands clean and dry, If you have any wound, cover it.
  • In case of an accident, burn or injury occurs, immediately communicate it to the teacher.
  • Remember where the medicine cabinet is located.
  • Keep the area clean and orderly.
  • Do not use any tools or machines without knowing its use and operation.
  • Handle with special care the fragile material, for example, glass.
  • Inform the teacher of broken or damaged material.
  • At the end of practice, clean and sort the material that has been used.

    • The team Bio-lignin places great emphasis on safety of synthetic biology. The instructors have particularly introduced importance of biological safety for us, they are going to help us a lot in safety stuff.
    Safety in the lab:
    Into the lab we have to follow a couple of rules to our safety, since clean our work area until scientific processes, like:

    Safety in the modeling:

    We decided to use parts already in the iGEM registry for our killswitch construct. We chose an oxygen repressible promoter(Part:BBa_K950002), a kozak sequence(BBa_J63003), analysis sequence(BBa_K809605), and a terminator(BBa_K801012) all optimized for yeast from the registry. A kozak sequence is a ribosome binding site for eukaryotic organisms. The lysis sequence is also known as Lambda Holin, a protein from a bacteriophage that has also been shown to be cytotoxic in eukaryotic cells (Agu 1753). We then pieced these parts together using 3A assembly. First we combined the promoter and kozak and the coding sequence and terminator, then all four to make a full device. After that we used standard assembly to insert the sequence into a yeast vector(pSB1C3), so it was ready to be transformed into our yeast chassis.



    Protocols




    Protocolo de Resuspensión de Biobricks:

  • Remember: Working with fire.

    • Guide the plate in the pot A1 in the part upper left and identify the pot you will use pointing with a marker. Using a 10 ul pipette, take a white tip and perforate the aluminum surface of biobrick. Discard the tip and with other, add 10 ul of MQ water at the pot, release the liquid and mix. It can be seen that the water is dyed because of the DNA pigmentation, wait for an intense color to take the liquid. To finish, place all the liquid as possible into a 0,6 ul Eppendorf tube and storage at 4°C for short-term use or at -20°C for a later use.




    Protocol of Cell-competents Bacterium:

  • Remember: working with fire.

    • In the Falcon tube with 5 ml bacterium E.Coli (cultivated) add 500ul, carry it sterile tube 1,5 ml o 2 ml, centrifuged a full speed for 5 minutes and discard the remained. Add 300ul of CaCl2 cold, resuspended and centrifuged again a full speed for 3 minutes, again remove remained and add 50 ul of CaCl2 cold and proceed to transform or save -20°C for later use.




    Transformation Protocol :

  • Remember: Working with fire and keep all materials on ice.

    • In a sterile tube of 1,5 or 2 ml add 50 ul of bacterium cells-competent, add 2 ul of DNA and mix with little hits, then let resting on ice for 20 a 30 minutes. Give a thermal shock of 42°C for one minute and put on ice for two minutes. Add 200 ul LB medium and incubate a 37°C for 20 a 30 minutes, finally grown in falcon tubes and incubate at 37°C.




    Cultive protocol:

  • Remember: Working with fire.

    • Complete the Falcón tubes with 5ml LB, then put apart the control tube and add 100ul of bacteria already transformed and 1ul of antibiotic (Clor or Amp) for every ml of LB, finally we incubate at 37°C.




    Protocols

    Add 1000ul (1ml) of bacteria cultivated into an Eppendorf tube and centrifuged a full speed for 5 minutes. Discard supernatant with help of pipette, leaving the sediment as dry as possible. Resuspend the pellet, add 250 ul of solution of resuspension with a pipette and resuspended, add 250ul of solution of lysis and mix the sample. Add 10ul of alkaline protease and mix four times, incubate for five minutes a room temperature. Add 350ul of neutralizing solution and mix four times, centrifuged a full speed for 10 minutes. Add 600 ul of supernatant in a column and this in a collector tube, centrifuged a full speed for a minute. Remove lysate and reintegrate the column in a collector tube. Add 750 ul of wash solution and centrifuged a full speed for a minute, discard lysate and reintegrate in the column. Add 250 ul of wash solution and centrifuged a full speed for 3 minutes. Transfer the column a un tube sterile 1,5 or 2ml, add MQ water and centrifuged a full speed for 3 minutes, finally keep the sample in a -20°C




    Double Digestion Protocol:

    In a sterile tube 0,6 ml add 10 ul of MQ water, add 5ul of sample, add 1ul of BSA (optional step), add 2 ul of buffer possessing more effectively, add 2ul of enzyme (1ul for every tube) and let rest for a hour at 37°C.




    Purify band protocol:

    Cut the band of the gel electrophoresis leaving in a sterile tube 1,5 or 2 ml and add 10 ul of solution of binding membrane for each 10 mg of a slice of gel. Let rest among 50° to 65°C until the gel is completely dissolved. Add 200 ul of sample in a column and this, in a collector tube, incubate a room temperature for a minute and centrifuged for two minutes. Discard the supernatant and reintegrate the tube, add 700ul of wash solution of membrane, centrifuged a full speed for two minutes, remove the lysate and reintegrate the column in the tube. Add 500ul of wash solution of membrane and centrifuged a full speed for six minutes, remove the lysate and transfer the column in a tube sterile 1,5 or 2 ml. Add 30ul of MQ water, centrifuged a full speed for two minutes, discard the column and finally keep the sample at 4°C or -20°C.




    Ligation Protocol:

    In a eppendorf tube add 3ul of MQ water, add 3ul of insert, add 1ul of vector, add 1ul of BSA (optional step), add 1ul of buffer ligase T4 and 1ul of ligase. Let rest for a hour at 37°C.




    Agarose Gel Electrophoresis Protocol:

    Weigh agarose powder and TAE buffer and add them to a flask. Melt the mixture in a lighter until the solution becomes clear and let the solution cool to about 40-50℃ and pour the solution into the gel casting tray with appreciate comb, let the gel cool until it is solid. Carefully pull out the comb. Place the gel in the electrophoresis chamber and add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel. Pipette DNA samples mixed with appreciate amount of loading buffer and dye into wells on the gel. Run the gel at 60V for about half an hour.