Team:Cornell/Week 13

From 2011.igem.org

Latest revision as of 17:23, 18 September 2011

August 28th - September 3rd

Sunday, August 28

Monday, August 29

Tuesday, August 30

Afternoon - Evening lab work done by: Claire Paduano, Youjin Cho, Maneesh Gupta, James Mathew, Charlie Chung

• Digestion Reactions for BioBrick Assembly

AviTagged RFP Insert

34.6µL ddH2O

7.9µL AviTagged RFP DNA (1 µg)

5µL 10x NEBuffer 3

0.5µL 100x BSA

1µL EcoRI

1µL PstI

50µL Total

AviTagged VioA Insert

28.3µL ddH2O

14.2µL AviTagged VioA DNA (1µg)

5µL 10x NEBuffer 3

0.5µL 100x BSA

1µL EcoRI

1µL PstI

50µL Total

AviTagged VioB Insert

26.7µL ddH2O

15.8µL AviTagged VioB DNA (1µg)

5µL 10x NEBuffer 3

0.5µL 100x BSA

1µL EcoRI

1µL PstI

50µL Total

Avi-Tagged VioE Insert

29.8µL ddH2O

11.7µL AviTagged VioE DNA (1µg)

5µL 10x NEBuffer 3

0.5µL 100x BSA

1µL EcoRI

1µL PstI

50µL Total

pSB1C3 Vector Backbone

40µL pSB1C3 DNA

5µL 10x NEBuffer 3

2.5µL ddH2O

0.5µL 100x BSA

1µL EcoRI

1µL PstI

50µL Total

• Dephosphorylation of pSB1C3 5' Ends via CIAP Treatment

- Please see the Protocol section for the procedure followed

• Gel Purification of Digestion Reaction Products via Electrophoresis

- Please see the Protocol section for the procedure followed

• Gel Extraction of DNA

- Please see the Protocol section for the procedure followed

- VioA and VioB genes migrated closely with their "native" pZE12 backbone, so initially both insert gene and backbone DNA bands were cut out

- After separating the two, a labeling mishap prevented the proper isolation of just the insert gene

- Thus, pZE12 backbone also underwent purification and ligation

• NanoDrop Spectrophometry for Purified DNA Quantification

- RFP = 16.8ng/µL

- VioA (gene?) = 33.7ng/µL

- VioA (pZE12?) = 17.6ng/µL

- VioB (gene?) = 27.8ng/µL

- VioB (pZE12?) = 18.6ng/µL

- VioE = 8.6ng/µL

- pSB1C3 backbone = 16.4ng/µL

• Ligation Reactions for BioBrick Inserts with iGEM Backbone

- Volumes are calculated as described in the Protocol section or, equivalently, with the Ligation Reaction Calculator spreadsheet

• RFP = 678bp
• VioA = 1257bp
• VioB = 2997bp
• VioE = 576bp
• pSB1C3 = 2070bp

AviTagged RFP + pSB1C3

6.1µL pSB1C3 backbone

5.8µL AviTagged RFP DNA

5.1µL ddH2O

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

AviTagged VioA (gene?) + pSB1C3

6.1µL pSB1C3 backbone

5.5µL ddH2O

5.4µL AviTagged VioA DNA (gene?)

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

AviTagged VioA (backbone?) + pSB1C3

Note: VioA (backbone?) treated as VioA gene insert in terms of #bp in Ligation Reaction Calculator

10.4µL AviTagged VioA DNA (backbone?)

6.1µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

0.6µL ddH2O

1µL T4 DNA ligase buffer

20µL Total

AviTagged VioB (gene?) + pSB1C3

12.2µL AviTagged VioB DNA (gene?)

4.8µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

AviTagged VioB (backbone?) + pSB1C3

Note: VioB (backbone?) treated as VioB gene insert in terms of #bp in Ligation Reaction Calculator

13.5µL AviTagged VioB DNA (backbone?)

3.5µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

AviTagged VioE + pSB1C3

9.7µL AviTagged VioE DNA

6.1µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

1.2µL ddH2O

1µL T4 DNA ligase

20µL Total

• Overnight benchtop incubation

Wednesday, August 31

Afternoon lab work done by: Youjin Cho

• Desalted the ligated samples from Tuesday and transformed them into MD37 electrocompetent cells using electroporation.
• After an hour of incubation, they were plated onto plate containing chloramphenicol.

Afternoon/Evening Lab work done by: Claire Paduano and Maneesh Gupta

• Looked at ATTO590 coated chip from Aug 27th under fluorescent microscope: channels still fluorescent

Photo to be posted soon

Streptavidin Coating and Fluorescent Probes

1) 45 min in 4% (by volume) MPTMS in ethanol

2) 20 min in 1mM GMBS

3) 45 min in 25ng/mL NeutrAvidin in PBS

• Streptavidin coating protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046

4) 20 min in fluorescent probe

• Incubated one chip with ATTO520, one in ATTO590

Flow Experiments

Details and photos to be posted soon

• ATTO520 gives signal under both Texas Red and GFPA filters
• ATTO590 gives signal under only Texas Red

- In future flow experimental design, keep in mind that we cannot differentiate between ATTO590 and 520 signal under Texas Red filter

Thursday, September 1

Morning lab work done by: Claire Paduano and Nancy Li

• Looked at ATTO590 coated chip from Aug 27th and chips from Aug31st under fluorescent microscope: channels still fluorescent
• Coated three chips with streptavidin

Streptavidin coating

• Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046

1) 45 min in 4% (by volume) MPTMS in ethanol

2) 20 min in 1mM GMBS

3) 45 min in 25ng/mL NeutrAvidin in PBS

Evening lab work done by: Maneesh Gupta

• Did continuous flow experiments to test resilience of biotin-avitin binding
• Used same exposure (83.33) in all images taken

Friday, September 2

Afternoon lab work done by: Charlie Chung

• Miniprep DNA purification of cultures containing pSB1C3 vector backbone with (RFP + AviTag), (VioA + AviTag), (VioB + AviTag), and (VioE + AviTag) inserts -- in preparation for iGEM BioBrick submission

Saturday, September 3

Afternoon lab work done by: Jim Mathew, Charlie Chung, Nancy Li

Quantification of Purified DNA Samples via NanoDrop Spectrophotometry

- All 260/280nm ratios were near 1.8, indicting pure DNA

- All inserts listed below are ligated with the AviTagged pSB1C3 vector backbone

RFP 1 -- 344.0ng/µL

RFP 2 -- 256.4ng/µL

RFP 3 -- 230.4ng/µL

VioA 1-1 -- 205.9ng/µL

VioA 1-2 -- 494.3ng/µL

VioA 1-3 -- 370.8ng/µL

VioA 2-1 -- 892.5ng/µL

VioA 2-2 -- 203.8ng/µL

VioA 2-3 -- 206.0ng/µL

VioB 1-1 -- 503.9ng/µL

VioB 1-2 -- 186.1ng/µL

VioB 1-3 -- 398.8ng/µL

VioB 2-1 -- 868.9ng/µL

VioB 2-2 -- 145.6ng/µL

VioB 2-3 -- 174.6ng/µL

VioE 1 -- 266.2ng/µL

VioE 2 -- 552.9ng/µL

VioE 3 -- 400.7ng/µL

• DpnI Digestion of PCR Reaction Product

- PCR with specially designed primers to delete the iGEM restriction enzyme cut sites, which may have been forming too much distance between the ribosome binding site and the start codon of our gene

- first half of the forward primer consists of the base pairs flanking the deletion region to the left, second half of the forward primer consists of the base pairs flanking the deletion region to the right

- when the primer anneals to the template, it is complementary to the nucleotides on either side of the deletion region and pinches them in, forcing the deletion region to loop out without a complementary strand

• Transformation via Electroporation

Note: Either the DH5α or MC4100 strain will have the degradation system of unmethylated DNA knocked out

- (RFP + AviTag) into DH5α and MC4100

- (VioA + AviTag) into DH5α and MC4100

- (VioB + AviTag) into DH5α and MC4100

- (VioE + AviTag) into DH5α and MC4100

• Plating of Transformed Bacteria

- Ampicillin-treated plates are incubating at 37°C in Weill Hall