Lab Write Up

Jan 18th: Workshop in Lethbridge. Took notes on procedures and definitions. We determined steps for our project: Transformation, grow overnight cultures, miniprep, restriction digest, ligation, transformation.

May 19: Grew JM109 cells overnight.

May 20: Transformed DNA into JM109 cells after making competent cells. Let grow overnight.

May 21: Miniprepped AmilCP and ECOS in JM109

May 22: Restriction digest of ECOS and AmilCP. Puc57 was cut with E and S. Amil CP in PSB1C3 was cut with E and X. cutsmart was used for the buffer.

May 25: Started JM109 cells. Grew for three hours while ligated parts together and transformed into JM109. OD was .51

May 26th: Running a gel to see our results. Labelling system as follows: Plate/tube #/U or B (uncolored colony or blue colony). Gel run as follows

Well 1) Amil CP 1
Well 2) Amil CP2
Well 3) 2/1/U
Well 4) 2/2/U
Well 5)1/1/U
Well 6) 1/2/U
Well 7)1/1/B
Well 8) 1/2/B
Well 9) 2/1/B
Well 10) 2/2/B

August 14th: We were unsure what happened at this point with the coloured and uncoloured culture so we decided to start from the beginning and see what happens. We grew overnight cultures of AmilCP and JM109. They grew well.

August 16th: Miniprepped the Amil CP and stored it in the freezer. Made plates with 4% alginate to make sure we can grow cultures on it.

August 17th: Mini prepped the old version of ECOS (We used ECOS from plates we had in the fridge furthermore referred to as old ECOS). Made competent cells from JM109 and transformed new ECOS (we transformed DNA from the freezer. This batch is refereed to furthermore as new ECOS). Cells were plated overnight. We also made alginate plates. One was a negative control and the other with JM109 cells.

August 18th: Took plates from incubator and started broth with new ECOS. Did restriction digest of AmilCP and old ECOS. We did two different trials. Note that we did both trials with old and new ECOS.

Trial A- puc57 is cut with x and p. was used for buffer. PSB1C3 was cut with E and P. 3.1 was used for the buffer.
Trial B- Puc57 was cut with E and S. Amil CP in PSB1C3 was cut with E and X. cutsmart was used for the buffer.

Labelling system as follows: type.tube# trial letter Ex. 1.1 A
1=AmilCP
2=ECOS old
3= ECOS new
4=PSB1C3
We then ligated our parts together for each of our respective trials. (Old ECOS)

Made competent cells- .5 OD and transformed our ligation into our cells. We ended up with 6 plates. 2 negative controls on amp plates and 4 plates on chlor- two plates from each trial with one 20 micro litre and one 200 micro litre of broth.

August 19th: Made LB cultures of JM109. Miniprepped New ECOS. Made Chlor. plates. Restriction digest of New ECOS. Ligation of New ECOS (using trial A and trial B from yesterday). Made competent cells- .4OD transformed them and grew overnight. We also ran a Gel of all our restriction digests.

ladder
1.1
1.2
1.3
1.4
2.1
2.2
3.1
3.2
3.1
3.2
4.2
Ladder

Our results were as followed:

As you can see the PSB1C3 was the farthest ahead which is what we want as it was the smallest part. All four of our new ECOS were triple banded. We are unsure why this is happening. As we were expecting two bands- one with ECOS and one with the backbone. But AmilCP is between the new ECOS and the PSB1C3 which is what was expected.

Aug 20th: New ECOS transformation results- Dark and light colonies on Trial B 200 micro litres and no growth on 20 micro litres. Some colonies on Trial A 200 micro litres and no growth on 20 micro litres.

Aug 26th: Grew overnight cultures of plates from New ECOS trial A and B.

Aug 27th: Labelling: Trial plate- colouring number (Originals: A1 A2 B1-D B2-D B1-L B2-L) L= light D=Dark (Example: B1-D1). We started a mini-prep but accidentally threw out supernatant we needed. Grew more LB cultures.

Aug 28th: Plate one: negative control. Plate two: 10 microlitres. Plate three: 100 Microlitres. Plate four: 250 microlitres.

tube one: negative control. tube two: 1 microlitre. tube three: 25 microlitres. Tube four: 500 microlitres.

We did a miniprep and restriction digest with ecoR1 and cutsmart on all trials. Ran gel with the following order.

Ladder
A1
A2
B1D
B2D
B1L
B2L
PCB1C3
Ladder

Results as follows:

Sept. 17th: In order to prepare for our "real life" experiments, we mixed 3.5mL of xylene for 350mL of soil. We then added the soil to our built prototype and added LB and cell cultures from yesterday to the chamber that holds ECOS. We then let it sit overnight, starting at 4 p.m. on the shaker table.