"

From 2011.igem.org

October 16th - October 22nd

Sunday, October 16

Morning lab work done by: Charlie Chung

Preparing 100mL Cultures of VioA, B, E and pZE12 Vector

• Inoculate two baffled flasks of (50mL LB + 50µL ampicillin) each with 1mL of yesterday's culture for a 1:50 dilution
• Shake at 37°C for 2.5 hours and check OD reading (if 0.05-0.08, then ready for IPTG-induction)
• OD after 2.5 hours = 0.1xx (VioA), 0.08 (VioB), 0.1xx (VioE) -- too dense again...

- Next time, check OD after 2 hours when doing a 1:50 subculture

• Induced with 5µL 1M IPTG for 0.1µM addition to a 50mL culture
• Shaking at room temperature for 20+ hours, after which we will pellet the cells for lysis and protein extraction

Night lab work done by: Nicholas Kramer

Making Microfluidic Chips

• Poured 55g of 10:1 PDMS over the molds and put in 60°C oven overnight

Monday, October 17

Morning lab work done by: Jim Mathew, Charlie Chung

• Submitted VioA, B, E and GFP for sequencing to confirm PCR deletion

Preparing VioA, B, E and pZE12 for Protein Extraction

• Spin down the 100mL of each culture into pellets (3700rpm for 30 min)

- Stored in -20°C fridge of Olin 301

Afternoon lab work done by: Maneesh Gupta and Claire Paduano

Making Microfluidic Chips

• Cut PDMS off of mold and bonded to glass slide with O2 plasma
• Tested resulting chips, 6 new chips made
• Poured 55g of 10:1 PDMS on molds and put in the 60°C oven overnight

Tuesday, October 18

Afternoon lab work done by: Bill Jo

Making Microfluidic Chips

• Cut out PDMS and bonded to glass slide using O2 plasma
• Tested resulting chips, 3 new chips made
• Poured 55g of 10:1 PDMS over mold and put in the 60°C oven overnight

Wednesday, October 19

Morning lab work done by: Bill Jo

Making Microfluidic Chips

• Cut out PDMS and bonded to glass slide using O2 plasma
• Tested resulting chips, 8 new chips

---

Afternoon lab work done by: Jim Mathew, Youjin Cho

Lysing GFP and pZE12 Control

• Lysed the GFP and pZE12 control using BugBuster
• After adding 5mL of BugBuster to each sample, shook in incubator for 20 minutes
• Spun the samples down for 20 minutes at 4°C at maximum speed

(Lysed samples of GFP and pZE12 control stored in 4°C)

Thursday, October 20

Friday, October 21

Afternoon lab work done by: Archana Rachakonda, Jim Mathew, Charlie Chung

• Spin down 1L cultures of newly transformed (because of sequencing results negative for PCR deletion) VioA, B, E and pZE12

- Stored in -20°C freezer of Olin 301

• PCR of GFP-AviTag-pZE12 to create final construct of Prefix-GFP-AviTag-Stop-Suffix
• Run on gel

- PCR failed: no bands of amplified product

• Redo PCR with two adjustments

(1) Lower annealing temperature from 53.2°C to 50.0°C

(2) Increase volume of GFP-AviTag-pZE12 template from 0.5µL to 1µL

Saturday, October 22

Lab work done at Olin Hall by: Jim Mathew, Charlie Chung

• Lysed cell pellets of VioA, B, E and empty pZE12 vector using BugBuster

- Note: VioB pellet is red. Acting on its substrate somewhere in the culture? Confirms active status of enzyme?

- Transferred lysate to teammates in Weill Hall running experiment of binding Vio enzymes to microfluidics chips and passing substrate (L-tryptophan) through

• Gel electrophoresis on PCR product to verify amplification

- PCR with two adjustments still failed: only bands of the ladder showed

Lab work done at Weill Hall by: Maneesh Gupta, Archana Rachakonda, Jim Mathew

• Ran full test of the Biofactory

- 4 groups in parallel with 3 coated chips in each group

- Enzymes were incubated in the chips for 1 hour with no flow. The enzyme solution was then replace with fresh enzyme solution and incubated again for 1 hr

- Flowed L-tryptophan solution through all 4 groups at 5µl/min for 6 hours

- Collected flow-through for analysis

• Group 1

VioA chip, VioB chip and VioE chip

• Group 2

VioA chip, Blank chip, VioE chip

• Group 3

VioA chip, VioB chip, Blank chip

• Group 4

Blank chip, Blank chip, Blank chip