Team:Caltech/Week 13

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:

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&degC
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:

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