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From 2011.igem.org

September 11th - September 17th

Sunday, September 11

~God bless America -- never forget~

Monday, September 12

Afternoon lab work done by: Nancy Li, Charlie Chung

• Prepared ten 3mL LB cultures (with 3µL carbenicillin) of colonies picked from plates dated Sept 3

- RFP + AviTagged pZE12 in DH5α

- VioA + AviTagged pZE12 in DH5α

- VioB + AviTagged pZE12 in DH5α

- VioE + AviTagged pZE12 in DH5α

- RFP + AviTagged pZE12 in MC4100

- VioA + AviTagged pZE12 in MC4100

- VioB + AviTagged pZE12 in MC4100

- VioE + AviTagged pZE12 in MC4100

- Control tube is just a colony with pZE12 (no insert gene)

- LB with carbenicillin (no inoculation with bacteria)

• Incubating at 37°C in the shaker at Weill Hall
• Cultures are to be lysed for release of natively biotinylated RFP and Vio enzymes in characterizing streptavidin-coated microfluidics chips
• Did not extensively search for L-tryptophan (starting substrate in the synthesis of prodeoxyviolacein) in the reagents shelf of our Weill Lab space, but does not appear to be in plain view (may be tucked away in an obscure corner?)

Tuesday, September 13

Afternoon lab work done by: Charlie Chung

Objective: Transfer yesterday's 3mL starter culture into today's larger scale culture (20mL LB + 20µL ampicillin)

• Autoclaved two 1L LB at 1:30pm (~1.5 hour cycle)
• Claire's batch of five autoclaved 250mL flasks are ready for use
• Prepared stock solution of 1000x ampicillin (100mg/mL)

- Weigh out 1g ampicillin sodium salt (Sigma)

- Dissolve in 10mL ddH2O

- Syringe filter (0.22µm membrane) the ampicillin solution

• Stored in the bottom shelf of Weill Lab refrigerator (it's in a 15mL conical -- will later aliquot into Eppendorfs)

Evening lab work done by: Claire Paduano

• Prepared cultures of AviTagged VioA, VioB, VioE and RFP in 20mL LB with 20µL ampicillin
• Left in 37°C shaker to culture

Wednesday, September 14

Thursday, September 15

Morning lab work done by: Claire Paduano, Jim Mathew

• DpnI Digestion of PCR Reaction Product

- Retried the PCR deletion method on AviTagged VioA, VioB, and VioE

AviTagged VioA

40.7μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.3μL dsDNA (20ng)

1.0μL PfuUltra High-Fidelity DNA polymerase

50.0μL Total

• 1µL fwd primer diluted in 5.3µL ddH2O before adding 1µl to reaction mixture
• 1µL rvs primer diluted in 5.6µL ddH2O before adding 1µl to reaction mixture

AviTagged VioB

40.6μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.4μL dsDNA (20ng)

1.0μL PfuUltra Hi-Fi DNA polymerase

50.0μL Total

• 1µL fwd primer diluted in 7.6µL ddH2O before adding 1µl to reaction mixture
• 1µL rvs primer diluted in 7.7µL ddH2O before adding 1µl to reaction mixture

AviTagged VioE

40.8μL ddH2O

5.0μL 10x PfuUltra buffer

1.0μL dNTPs

1.0μL diluted forward primer (125ng)

1.0μL diluted reverse primer (125ng)

0.2μL dsDNA (20ng)

1.0μL PfuUltra Hi-Fi DNA polymerase

50.0μL Total

• 1µL fwd primer diluted in 6.2µL ddH2O before adding 1µl to reaction mixture
• 1µL rvs primer diluted in 5.6µL ddH2O before adding 1µl to reaction mixture

Run PCR protocol as listed under 'PCR Deletion' in protocols sections

Note: fwd and rvs primer dilution calculations based on our particular primer MWs and stock elution volumes

• Transformation via Electroporation

• Add 1.0µL DpnI to each PCR reaction
• Incubate in thermocycler at 37°C for 1 hour
• After incubation, transform PCR product into electrocompetent cells

- (VioA + AviTag) into DH5α

- (VioB + AviTag) into DH5α

- (VioE + AviTag) into DH5α

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Afternoon lab work done by: Youjin Cho, Charlie Chung

• Gel Purification of Digested Light-lysis Gene Insert

- After NanoDrop DNA quantification, 26ng/µL

• Ligation of Light-lysis Gene Insert with pSB1C3 iGEM Backbone

9µL light-lysis (insert) DNA

8µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

• Control Ligation Setup

9µL ddH2O

8µL pSB1C3 backbone

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

• Plated transformed products of PCR deletion technique on VioA, VioB, and VioE using ampicillin-treated agar plates

Afternoon microfluidics work done by: Maneesh Gupta

• Tested effect of fluorescein with lysate on streptavidin-biotin binding to see if lysate interferes with their affinity

- Used 5:1 ratio of lysate to fluorescein

- Chips used in the experiment were coated yesterday (streptavidin is 1 day old)

- Flow rate = 1µL/min for 50 minutes

- Tested chips "Aaaaahhh" and "Chippy". Test chip was "Aaaahhh" which contained lysate + fluorescein. "Chippy"  :: contained lysate. Stored both chips in fridge with DI water. Pictures taken with chips filled in air.

• Tested chip "stardust" with fluorescein two days after it was coated with streptavidin. Stored in DIwater in fridge.

- Flow rate = 5µl/min for 20min. pics taken in air.

Friday, September 16

Morning lab work done by: Youjin Cho, Archana Rachakonda

• Desalted the light-lysis + pSB1C3 ligation and control on a membrane for 15 minutes.
• Transformed the desalted samples into DH5α cell lines through electroporation and put them into 37°C shaker for 90 minutes.

Afternoon lab work done by: Jim Mathew, Claire Paduano, Charlie Chung

• Plates for transformation products of PCR-deletion technique did not grow colonies

- Arced electroporation & transport of plates from Weill to Olin in 40°C weather immediately after plating

- Will retry the PCR tomorrow (Saturday, September 17)

• Plated the (light-lysis + pSB1C3) and control (pSB1C3 only) transformation products on agar treated with chloramphenicol

- Incubating at 37°C in Olin 303

Saturday, September 17

Morning lab work done by: Jim Mathew, Claire Paduano

• Frustrating News -- Checking for Growth of Transformed Products

- Very few colonies of (light-lysis + pSB1C3) grew

- Equal or greater number of colonies grew on the Control (pSB1C3 only) plate