Team:Cornell/Week 20

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Latest revision as of 18:02, 28 October 2011

Results | Protocol | Notebook | Parts Submitted

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

@@ Line 19: / Line 19: @@
 Night lab work done by: Nicholas Kramer
 :'''Making Microfluidic Chips'''
-::*Poured 55g of 10:1 PDMS over the molds and put in 60 degree Celsius oven overnight
+::*Poured 55g of 10:1 PDMS over the molds and put in 60°C oven overnight
 ==Monday, October 17==
@@ Line 33: / Line 33: @@
 ::*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 degree Celsius oven overnight
+::*Poured 55g of 10:1 PDMS on molds and put in the 60°C oven overnight
 ==Tuesday, October 18==
@@ Line 40: / Line 40: @@
 ::*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 degree Celsius oven overnight
+::*Poured 55g of 10:1 PDMS over mold and put in the 60°C oven overnight
 ==Wednesday, October 19==
@@ Line 47: / Line 47: @@
 ::*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
+::- '''''<u>Note</u>''''': 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