Team:Cornell/Week 16

From 2011.igem.org

Revision as of 01:16, 29 September 2011

September 18th - September 24th

Sunday, September 18

Monday, September 19

Lab work done by: Jim Mathew, Youjin Cho, Charlie Chung

Background

• The PCR site-directed mutagenesis done earlier today should ideally enhance protein expression of VioA, VioB, VioE, and RFP by deleting excessive nucleotides between the ribosome binding site and the ATG start codon in the original DNA sequences

Work Accomplished

• PCR procedure for deletion technique
• Using 1µl of PCR product of VioA, VioB, VioE and RFP, transformed them into DH5α electrocompetent cells via electroporation
• Put the transformed samples in 37°C shaker for an hour
• Plated the transformed products of VioA, VioB, VioE, and RFP on agar plates treated with ampicillin or carbenicillin (two are on amp; other two are on carb)
• Incubating at 37°C in Olin 303

Tuesday, September 20

Afternoon lab work done by: Charlie Chung

• VioA, VioB, and VioE plates = lots of bacterial colonies, high-density growth

- Picked three colonies from each plate and started up (3mL LB + 3µL carbenicillin) cultures

• VioA-1,2,3
• VioB-1,2,3
• VioE-1,2,3

- Incubating at 37°C in Olin 304

• RFP plate = ~5 colonies

- Picked two colonies and started up (3mL LB + 3µL carbenicillin) cultures

• RFP-1,2

- Incubating at 37°C in Olin 304

Wednesday, September 21

Morning lab work done by: Charlie Chung

• Transferred 500µL from each culture tube to 20mL (LB + carbenicillin) subcultures

- Shaking at 37°C in Olin 304

• Miniprep the remaining cultures of VioA-1,2,3 ; VioB-1,2,3 ; VioE-1,2,3 ; RFP-1,2

- Because of time constraint, was not able to quantify the DNA concentration

- Sitting in the freezer of Olin 301

Afternoon lab work done by: Nancy Li, Youjin Cho

• After 5 hours of subculturing, transferred all the samples into corresponding 50mL tubes and spun them down at 4500rpm for 30 minutes
• Poured off the supernatant and stored the pellets at -20°C for future use

Thursday, September 22

Afternoon lab work done by: Charlie Chung

• Quantified the Miniprep-purified products with NanoDrop

- VioA-1 = 68ng/µL

- VioA-2 = 60.2ng/µL

- VioA-3 = 88.1ng/µL

- VioB-1 = 117.6ng/µL

- VioB-2 = 89ng/µL

- VioB-3 = 133.6ng/µL

- VioE-1 = 80.5ng/µL

- VioE-2 = 73.6ng/µL

- VioE-3 = 77.4ng/µL

- RFP-1 = 84.4ng/µL

- RFP-2 = 56.5ng/µL

• Prepared samples for sequencing submission

Order Number:10257313

VioA-1

15µL VioA-1+pZE12 (1µg)

2µL ddH2O

1µL pZE12 forward primer

18µL Total

VioA-2

17µL VioA-2+pZE12 (1µg)

1µL pZE12 forward primer

18µL Total

VioA-3

12µL VioA-3+pZE12 (1µg)

5µL ddH2O

1µL pZE12 forward primer

18µL Total

VioB-1

9µL VioB-1+pZE12 (1µg)

8µL ddH2O

1µL pZE12 forward primer

18µL Total

VioB-2

12µL VioB-2+pZE12 (1µg)

5µL ddH2O

1µL pZE12 forward primer

18µL Total

VioB-3

9µL ddH2O

8µL VioB-3+pZE12 (1µg)

1µL pZE12 forward primer

18µL Total

VioE-1

13µL VioE-1+pZE12 (1µg)

4µL ddH2O

1µL pZE12 forward primer

18µL Total

VioE-2

14µL VioE-2+pZE12 (1µg)

3µL ddH2O

1µL pZE12 forward primer

18µL Total

VioE-3

13µL VioE-3+pZE12 (1µg)

4µL ddH2O

1µL pZE12 forward primer

18µL Total

RFP-1

12µL RFP-1+pZE12 (1µg)

5µL ddH2O

1µL pZE12 forward primer

18µL Total

RFP-2

17µL RFP-2+pZE12 (1µg)

1µL pZE12 forward primer

18µL Total

Friday, September 23

Afternoon lab work done by: Charlie Chung

• Picked two colonies each from Sean's GFP+AviTag+pZE12 and RFP+AviTag+pZE12 plates and started (3mL LB + 3µL ampicillin) cultures

- Shaking at 37°C in Weill Hall

Saturday, September 24

Microfluidics work done by: Maneesh Gupta Afternoon-Evening lab work done by: Jim Mathew, Nancy Li, Charlie Chung, Claire Paduano

Preparation of iGEM's pSB1C3 Backbone

• PCR
• Digest with EcoRI and PstI
• CIAP treatment to prevent self-ligation

Preparation of Light-Induced Lysis Kit Insert

• 3.7ng/µL, 260/230 = 0.05

Ligation of Light-Induced Lysis Kit with iGEM's pSB1C3 Backbone

Insert Positive

13.3µL Light-Induced Lysis Kit insert (digested with EcoRI and PstI)

3.7µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

Control

13.3µL ddH2O

3.7µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

• Incubating overnight in 16°C water bath of Olin 304

Preparation of GFP+AviTag Insert

• PCR of Sean's GFP+AviTag+pZE12 construct

Note: Thermocycler in our Weill Hall lab does not seem to be cooling down quickly enough in between cycles. Retried PCR reaction in Olin Hall lab. Tube got warped again.

- Primers are designed to amplify ("lift off") only the GFP+AviTag and simultaneously add the iGEM prefix and suffix

• Ran PCR reaction product on an agarose gel to check for amplification and to purify the DNA

- Observed bright bands with migration rate matching 700bp of the ladder

• Gel purification and DNA quantification via NanoDrop spectrophotometry

- [GFP+AviTag] = 82 ng/µL

- 260/230 ratio was below 2.0-2.2, suggesting contaminants are still present in gel-purified product

• Digest the GFP+AviTag insert to create sticky ends that match iGEM's pSB1C3 backbone

30.3µL ddH2O

12.2µL GFP+AviTag (1 µg)

5µL 10x NEBuffer 4

0.5µL 100x BSA

1µL EcoRI

1µL PstI-HF

50µL Total

• PCR Clean Up of the digestion product of GFP+AviTag

Ligation of GFP+AviTag with iGEM's pSB1C3 Backbone

Insert Positive

9.3µL pSB1C3 backbone (digested with EcoRI and PstI & CIAP-treated)

4.2µL GFP+AviTag insert (digested with EcoRI and PstI)

3.5µL ddH2O

2µL 10x T4 DNA ligase buffer

1µL T4 DNA ligase

20µL Total

Control

• The control reaction tube from the light sensor ligation serves as the control for this ligation reaction as well
• Incubating overnight in 16°C water bath of Olin 304