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From 2011.igem.org

July 31st - August 6th

Sunday, July 31

Monday, August 1

Lab work done by: Jim Mathew

• Sent VioE and AviTag + backbone in for sequencing

Tuesday, August 2

Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo

Microfluidics

• Dr. Archer showed us how to use the clean room
• Made a SU-8 master of our device
• Poured PDMS and baked overnight at 60°C
• Followed protocol that is used by Biomedical Engineering lab class and is provided by Dr. Archer

Wednesday, August 3

Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo

Microfluidics

• Dr. Archer showed us how to cut out PDMS devices and seal to clean glass slides using plasma cleaner.
• Tested chips for blockages in flow

Three working devices, one with complete blockage in the channel

• Poured more PDMS and baked overnight at 60°C

Used protocol provided by Dr. Archer from Biomedical Engineering lab class

Thursday, August 4

Morning lab work done by: Jim Mathew

Objective

Prepare Avi-Tagged pZE12 vector backbone for GFP, RFP, and vioE gene inserts

Digestion Setup in Duplicate

32.6μL H2O

9.9μL Avi-Tagged pZE12 vector backbone (2μg)

5μL 10x NEBuffer 4

0.5μL 100x BSA

1μL KpnI-HF (HF = high fidelity)

1μL SphI-HF

50μL Total

Incubate in 37°C water bath for 2 hours

Dephosphorylation of 5' Ends of Vector Backbone

1. Add 1μL of Calf Intestine Alkaline Phosphatase (CIAP) to digested vector backbone in order to prevent self-ligation without gene insert included
2. Incubate at 50°C for 5 minutes
3. Run vector backbone through agarose gel electrophoresis

Gel Extraction and Purification of Digested Backbone

• Followed standard Qiagen Gel Extraction protocol for pZE12 vector backbone that is now Avi-Tagged and digested with KpnI and SphI
• NanoDrop spectrophotometry on the duplicate samples reported 12.7ng/μL and 14.2ng/μL

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Afternoon lab work done by: Claire Paduano, Charlie Chung

Objective

Ligation of GFP, RFP, and vioE genes with Avi-Tagged pZE12 backbone

Ligation

• GFP + Avi-Tagged Backbone

10.6μL Avi-Tagged pZE12 vector backbone

3.6μL GFP

2.8μL H2O

2.0μL T4 DNA Ligase Buffer

1.0μL T4 DNA Ligase

20μL Total

• RFP + Avi-Tagged Backbone

10.3μL Avi-Tagged pZE12 vector backbone

6.7μL RFP

2.0μL T4 DNA Ligase Buffer

1.0μL T4 DNA Ligase

20μL Total

• vioE + Avi-Tagged Backbone

10.6μL Avi-Tagged pZE12 vector backbone

4.1μL vioE

2.3μL H2O

2.0μL T4 DNA Ligase Buffer

1.0μL T4 DNA Ligase

20μL Total

In all above reactions, volume of plasmid vector added corresponds with 100ng backbone.

Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs.

• Same volume of vector backbone, buffer, ligase
• Volumes of all inserts (i.e. gene) go toward volume of H2O

After ligation setup, incubate in 16°C waterbath overnight.

Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocol page under the Multimedia tab) that is based on 100ng of vector backbone and a 3:1 molar ratio of insert:vector.

Reference for Inserts and Vector Backbone

RFP gene - 678bp

GFP gene - 717bp

vioE gene - 576bp

pZE12 vector - 2340bp

Friday, August 5

Lab work done by: Youjin Cho and Claire Paduano

Objective

Transform ligation of GFP/RFP/vioE + Avi-Tagged backbone

1. Desalted overnight ligation
2. Transformed the samples into DH5α electrocompetent cells

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Group Meeting

• Just finished transforming GFP/RFP/vioE + Avi-Tag + pZE12 backbone
• If the vioE gene successfully inserts and ligates with the Avi-Tagged backbone, then we can ligate in vioA and vioB this weekend
• Goal: to treat PDMS devices to coat with streptavidin and bind Avi-Tagged GFP and RFP to device

Saturday, August 6

Lab work done by: Youjin Cho, Charlie Chung

• Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol)

NOTE: White masses were floating in bacteria culture -- potential contamination?

• NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product

RFP (Colony 1) + Avi-Tagged pZE12 vector backbone = 53.2ng/µL

RFP (Colony 2) + Avi-Tagged pZE12 vector backbone = 63.4ng/µL

RFP (Colony 3) + Avi-Tagged pZE12 vector backbone = 76.6ng/µL

GFP (Colony 1) + Avi-Tagged pZE12 vector backbone = 65.7ng/µL

GFP (Colony 2) + Avi-Tagged pZE12 vector backbone = 65.5ng/µL

GFP (Colony 3) + Avi-Tagged pZE12 vector backbone = 46.6ng/µL

vioE (Colony 1) did not grow in its culture tube

vioE (Colony 2) + Avi-Tagged pZE12 vector backbone = 53.8ng/µL

DNA from vioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed.