This homework assignment is focused on learning how to walk through a biological synthesis project, developing lab skills, understanding basic synthetic biology terminology, and reproducing a paper on synthesizing a ladder.

I began this week by reading the paper and, partially still confused, trudging into lab and getting to work.

To answer the homework questions:

    Software: Online viewer on addgene
      pstI: 500, 2,000, 1,000, 700, 800, 900, in that order. (7 cuts)
        ecoRV: 500, 1,000, 1,500, 2,000, in that order (5 cuts)
          ecoRV: Cut is after (GAT) in (GATATC). PstI: Cut is after CTGC in CTGCAG
            As far as I can tell, the only genes encoded onto these pPSU plasmids are ladder fragments, EcoRV and PstI of various ladder lengths.
              Since these are high copy, we expect hundreds of copies (300-500) per cell.
                By contrast, pBR322 is low copy number, and we'd only expect around 10-20 copies of the plasmid in there; it reproduces much slower.

                I began the lab by collecting the pPSU1 and pPSU2 α cultures:

                and adding, in order, the Zymopure P1:

                The Zymopure P2:

                And the Zymopure P3:

                These chemicals lyse the cell in a standardized procedures, breaking down the cell membrane (and, probably, other organelles), releasing the plasmids into the mixture. We only vortex once, early on while the cell is mostly intact - vortexing in later steps can damage the plasmids, destroying the genetic material, defeating the purpose.

                After that, I incubated the cells on ice for 5 minutes. I'm not actually completely sure why this is, I need to look this up and come back to that.

                After this, the supernatant (separated fluid, which, in our case, contains the released plasmids) was removed and mixed with a buffer, to keep the pH at a workable range.

                and then filtered and centrifuged,

                removing everything but the solid genetic material.

                And then this is the moment that everything went wrong.

                The last steps are to wash the genetic material of any other debris (biological or otherwise) that may have gotten trapped there so far. There is a standard "ZyMO" process for doing this, involving using one wash once, and then another wash twice. Unfortunately, we accidentally picked up the wrong bottle and did wash 2 first, completely destroying our genetic material.

                So even though this looks like good filtering that came from the wash:

                With good centrifuging to flow the wash through (which was then removed each time before adding more wash material)

                It was all for naught. The resulting spectrums and yield were all nonsense. Out of all the nanodropped materials of our bench, this was our best, with a measly yield of 46.3 ng / uL for pPSU2 (and 70.5 for pPSU1, not shown). Washing out of order really does a number on your genetic material...

                Instead, this procedure was all repeated the next day. The final steps after washing was simply to add it to DNA/RNA-free water (don't want to contaminate our genetic material with other genetic material; apparently most water from the tap has some genetic material in it):

                And incubate it (again, have to figure out why at this point as there's no enzyme). After redoing the whole experiment as a group up to this point, the yields and spectrum made much more sense:

                That's a yield of 154.1 ng / uL for pPSU1 and 1,692.6 ng / uL for pPSU2, and a much more conforming curve to go along with it.

                Hooray! On to gel electrophoresizing (is that the right verb form?).

                In order to prepare our materials for gel electrophoresis, we have to take our 25 uL of each of our larger quantity genetic material (pPSU2) and dilute it to 40 uL. That's 5 uL of material for each of the 8 people at the bench that we can use for our downstream experiments. Actually, we needed more than this, but we messed up, so we have no control for pPSU2 ( :( ). For pPSU1, the yield was a bit smaller, so we used all of the material without diluting.

                In order to dilute to 40 uL, we performed thefollowing calculation:

                We needed 40 uL at 100 ng/uL. That means we needed 4000 ng total. Since we had material 1692.6 ng / uL, we added 4,000 ng / 1,692.6 ng/uL = around 2.36 uL of our pPSU2 mixture, and diluted it with gene-free water (around 37.64 uL)

                Finally, we created our mixtures of pPSU1 and pPSU2 mixed with the appropriate buffers and enzymes (one vial of EcoRV-HF, and one of PSTI, at 50 uL total. We added 1 ug of each pPSU1 and pPSU2, meaning we needed 1,000 / 54 = approx 6.5 uL of pPSU1, and 10 uL of pPSU2. The rest of the volumes followed the specified online amounts (5uL buffer, 2uL enzyme, the remainder water). Tada! Our specimens:

                The remainder involved thermocycling our material (to incubate it get the cutting enzymes working faster), setting up our gel electrophoresis kit, mixing references, our specimens, and the control (only one because we didn't make enough for pPSU2) with loading dye, and depositing them in the gel mold. Because I was loading the gel and I was the only one there when starting the loading, I did everyone else's specimens but mine - I felt weird leaving someone else out arbitrarily. Results from our gel electrophoresis can be seen on our group page (TOOD).

                On the far left is the 100bp reference ladder; on the far right, the 1kb pair reference ladder. The second from the left is the control - the plasmid doesn't separate because there's no enzyme. After that, it's lanes of EcoRV, PstI, repeated three times. Most of the results look good except for Mina's EcoRV-HF. I'm not yet sure why that is. Lane four has some "smudging" because I accidentally locally punctured some of the gel in that area.