September 4
Overnights of: K123001 (bisdB) x2, pNT001 (construct vector) x2, and pSB3k3(BioBrick submission vector) x3
September 5
Miniprepped overnights of K123001, pNT001, and pSB3k3
PCR assembly of K123001 with prefix-promoter and terminator-suffix ultrimers to make bisdB construct
Gel of PCR assembly product
Overnights of J23119-lacZ-MG-1655 for x-gal analysis
Results
Miniprep concentrations:
| Miniprep |
Nanodrop conc. (ng/uL) |
| pNT001-a |
41 |
| pNT001-b |
83.5 |
| K123001-a |
101.8 |
| K123001-b |
68.0 |
| pSB3k3-a |
69.3 |
| pSB3k3-b |
83.4 |
| I372017 |
0 |
| pSB3k3-c |
54.1 |
Gel reveals correct sized product of ~1.5kbp
|
September 6
Assembly of bisdB pcr'ed construct into pNT001 (with bisdA) and pSB3k3 (BioBrick submission vector, with unwanted RFP)
Plate all assembled & transformed constructs and controls
Grow overnights of J23119-lacZ-MG-1655 from two different plate sources and one glycerol stock
September 7
Plated the rest of transformed pSB3k3-bisdB
Add the J23119-lacZ-MG-1655 to autoclaved LB+glass beads to incubate biofilm at 37°C
Performed qualitative X-gal experiment to pick best J23119-lacZ-MG-1655 overnight (turned most blue in least time)
Restreak pNT001-bisdB colonies onto another plate for clearer single colonies
Results
Plate results:
| Plate |
Number of colonies |
| pNT001-bisdB |
15 |
| pSB3k3-bisdB |
0 |
| pSB3k3-control #1 (just digestion) |
20, all red |
| pSB3k3-control #2 (digestion + ligation) |
6, one white |
| pSB3k3-control #3 (digest, dephosphorylate, ligate) |
0 |
September 8
Overnights of all pNT001-bisdB and pSB3k3-bisdB colonies for sequencing tomorrow
Transformed DDT-pET protein overexpression vector into BL21D3 strain
Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment
PCR amplification for BisdB parts mix using phusion:
Volume: 50 ul
- Temp 98°C for 30 seconds
- Temp 98°C for 10 seconds
- Temp 55°C for 20 seconds
- Temp 72°C for 30 seconds
- Go to 2, 9
- Temp 98°C for 10 seconds
- Temp 65°C for 20 seconds
- Temp 72°C for 30 seconds
- Go to 6, 19
- Temp 79°C for 3 minutes
- Temp 4°C forever
- END
Results
Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony
September 9
Minipreps and quikchange kit
Quikchange kit pcr for a 25 ul reaction:
- 2.5 ul of 10x buffer
- 5 ul of plasmid
- 1 ul of primer A
- 1 ul of primer B
- 1 ul of dNTP mix
- 1 ul of lightning
- 0.75 ul of Quik Solution
- 12.75 ul of water
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