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From 2011.igem.org

August 7th - August 13th

Sunday, August 7

Morning lab work done by: Youjin Cho, Claire Paduano

Objective

Prepare VioE in AviTagged pZE12 vector backbone for VioA and VioB gene inserts

Digestion Setup

23.65μL H2O

18.6μL VioE in AviTagged pZE12 vector backbone (1μg)

5μL 10x NEBuffer 2

0.5μL 100x BSA

1.25μL KpnI

1μL HindIII

50μL Total

KpnI has only 75% efficiency in Buffer 2, so added 0.25μL more KpnI to digestion mixture

Incubate in 37°C water bath for 2 hours

Afternoon lab work done by: Jim Mathew, Charlie Chung

Objective

Ligate VioA and VioB genes onto their own pZE12 vector backbone, in place of the VioE that is now digested out

Ligation Reaction Setup

• VioA + AviTagged pZE12 vector backbone

11.4μL pZE12 backbone (cut with KpnI & HindIII; former VioE gene digested out)

3.3μL VioA gene insert (cut with KpnI & HindIII)

2.3μL H2O

2μL 10x T4 DNA ligase buffer

1μL T4 DNA ligase

20μL Total

• VioB + AviTagged pZE12 vector backbone

9μL VioB gene insert (cut with KpnI & HindIII)

8μL pZE12 backbone (cut with KpnI & HindIII; former VioE gene digested out)

2μL 10x T4 DNA ligase buffer

1μL T4 DNA ligase

20μL Total

• Control Ligation for VioA and VioB Gene Inserts

11.4μL pZE12 backbone (cut with KpnI & HindIII; former VioE gene digested out)

5.6μL H2O (gene insert volumes go toward water volume)

2μL 10x T4 DNA ligase buffer

1μL T4 DNA ligase

20μL Total

Incubate all ligation reaction constructs overnight in 16°C water bath

Evening lab work done by: Charlie Chung

Objective

• Because previous DH5α colonies transformed with (VioE + AviTagged backbone) are very small, in addition to one failed 5mL culture and one discarded Miniprep sample, incubated plate for four more hours
• Picked three new colonies. Labeled as "VioE + AviTag + BB #4, 5, 6"
• 5mL LB cultures with 5μL ampicillin incubated overnight in 37°C shaker of Room 304

Monday, August 8

Morning lab work done by: Youjin Cho, James Mathew

Objective

• Transform the overnight ligation of VioA/VioB + AviTag + pZE12
• Send the Miniprepped samples (GFP, RFP, VioE) from Saturday in for sequencing
• Miniprep the additional VioE samples that were grown overnight

Transformation

• Desalted the ligation reaction samples (VioA/VioB + AviTag + pZE12) on a membrane for 15 minutes
• After adding the samples into DH5α cell lines, electroporated them and put into shaker for 45 minutes
• The samples were plated on the plate with ampicillin

Sequencing

• Using the reverse primer dilution, the samples were sent in for sequencing

GFP + AviTag + pZE12 - 1

GFP + AviTag + pZE12 - 2

GFP + AviTag + pZE12 - 3

RFP + AviTag + pZE12 - 1

RFP + AviTag + pZE12 - 2

RFP + AviTag + pZE12 - 3

VioE + AviTag + pZE12 - 2

Miniprepping the VioE samples

• The samples were Miniprepped using standard Qiagen Miniprep protocol

VioE + AviTag + pZE12 - 4

VioE + AviTag + pZE12 - 5

VioE + AviTag + pZE12 - 6

Evening lab work done by: Charlie Chung

• Checked to see if colonies were ready to pick from transformed plates of (VioA + AviTagged pZE12 backbone) and (VioB + AviTagged pZE12 backbone)

• VioA plate is showing early signs of a good number of colonies. Too small for picking, however
• VioB and Control plates look pretty empty (but just may not have grown up enough to tell)

• Will be best if colonies were picked Tuesday (tomorrow) morning. Please find the 3 plates still in the 37°C incubator

Tuesday, August 9

Evening lab work done by: Youjin Cho

• Picked colonies from transformed plates of (VioA + AviTagged pZE12 backbone) and (VioB + AviTagged pZE12 backbone).

VioA 1,2,3

VioB 1,2,3

• Put them into the 37°C shaker around 5:30pm

Wednesday, August 10

Morning lab work done by: Youjin Cho, Alyssa Henning

Objective

• Miniprep the samples of VioA/VioB + AviTag + pZE12 backbone and measure their concentrations
• Submit the Miniprepped samples for sequencing
• Since the sequencing results showed that GFP did not work, run a PCR reaction for more GFP

DNA Purification of VioA, VioB Samples

• The samples were Miniprepped using standard Qiagen Miniprep protocol

VioA + AviTag + pZE12 - 1 = 67.6 ng/µL

VioA + AviTag + pZE12 - 2 = 70.6 ng/µL

VioA + AviTag + pZE12 - 3 = 71.4 ng/µL

VioB + AviTag + pZE12 - 1 = 66.0 ng/µL

VioB + AviTag + pZE12 - 2 = 70.6 ng/µL

VioB + AviTag + pZE12 - 3 = 75.9 ng/µL

Sequencing

• The Miniprepped samples were prepared for sequencing using the diluted reverse pZE12 primer

Order Number: 10254703

VioA-1

VioA-2

VioA-3

VioB-1

VioB-2

VioB-3

PCR for GFP

• Since the result from the sequencing showed that GFP did not work, ran a PCR reaction using the original template of GFP in pZE12
• Ran two reactions and labeled them 1,2 GFP iGEM 8/10

1 GFP = the sample from May 5 of GFP on pZE12 with concentration of 50.6 ng/µL

2 GFP = the sample missing the date with concentration of 20.2 ng/µL

• The PCR was ran with melting temperature of 54.2°C for 1 minute

Thursday, August 11

Morning lab work at Olin Hall done by: Youjin Cho

Objective

• Purify the GFP that was PCRed off the GFP with pZE12 backbone
• Digest 1µg of the DNA of GFP and AviTag with pZE12 backbone

PCR Purification

• The GFP PCR samples were purified using the Qiagen Kit
• Using NanoDrop the two samples of GFP were quantified:

GFP-1 = 206.4ng/ul

GFP-2 = 159.2ng/ul

Digestion Setup

GFP-1

37.2μL H2O

4.84μL GFP sample-1 (1μg)

5μL 10x NEBuffer 4

1μL KpnI-HF (HF = high fidelity)

1μL SphI-HF

50μL Total

GFP-2

35.7μL H2O

6.28μL AviTagged pZE12 vector backbone (1μg)

5μL 10x NEBuffer 4

1μL KpnI-HF (HF = high fidelity)

1μL SphI-HF

50μL Total

AviTagged pZE12 backbone

32.4μL H2O

9.59μL AviTagged pZE12 vector backbone (1μg)

5μL 10x NEBuffer 4

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

• Add 1μL of Calf Intestine Alkaline Phosphatase (CIAP) to digested vector backbone in order to prevent self-ligation without gene insert included
• Incubate at 50°C for 5 minutes
• After adding 6x loading buffer, run samples through agarose gel electrophoresis

Gel Extraction and Purification of Digested Backbone

• Followed standard Qiagen Gel Extraction protocol for samples from digestion reaction
• NanoDrop spectrophotometry on the duplicate samples reported:

GFP-1 = 10.3 ng/μl

GFP-2 = 7.5 ng/μl

AviTag + backbone = 11.6ng/μl

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Weill Hall lab work done by: Charlie Chung, Claire Paduano, Bill Jo, Nick Kramer, Youjin Cho

Objective

• Coat microfluidic device with steptavidin

Diluting Chemicals

• 4% MPTMS in ethanol (MPTMS = 3-mercaptopropyl trimethoxysilane)

4mL MPTMS

96mL EtOH

• 1mM GMBS in ethanol

0.014gGMBS (280.2g/mol)(GMBS = N-g-maleimidobutyryloxy succinimide ester)

50mL EtOH

• 25ng/mL NeutrAvidin in PBS - serial dilutions

Dilute 0.25mg in 10mL PBS = 0.025mg/mL

Take 1mL of first dilution and bring to 10mL = 2.5µg/mL

Take 1mL of second dilution and bring to 100mL = 25ng/mL

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

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Evening lab work done by: Charlie Chung

• Started three additional cultures of (RFP + AviTagged pZE12 backbone) in 3mL LB with 3μL ampicillin

• Picked Colonies #4, 5, and 6 from (RFP + AviTag + pZE12) plate

- Because sequencing results are positive, assuming all colonies are also successful clones

• Incubating overnight in 37°C shaker of Room 304

To be used for tomorrow (Fri, 8/12) when lysing bacteria and releasing RFP-containing lysate into microfluidic channel for observation under fluorescent microscope

• Started three additional cultures of (AviTagged pZE12 backbone) in 3mL LB with 3μL ampicillin

• Picked Colonies #4, 5, and 6 from (AviTag + pZE12) plate

- Because sequencing results are positive, assuming all colonies are also successful clones

• Incubating overnight in 37°C shaker of Room 304

Friday, August 12

Team Meeting

• Checked the sequencing for VioA and VioB -- VioA worked, but VioB didn't

- Need to PCR off the VioB gene and repeat ligation with AviTagged backbone

• Need to figure out the flow rate that we will use for the microfluidics device
• Ask Dr. Archer about the protocol for taking picture/video with the microscope

Lab Work for This Week

Microfluidics

1. Need to induce the RFP cultures overnight (Friday evening)
2. Lyse the cells and run through the device to see if works (Saturday afternoon)

Lab Work

1. Transform the ligation of GFP + AviTag + pZE12 backbone and incubate overnight (Friday evening)
2. Store the plates away in 4°C and pick colonies from them late at night (Saturday)
3. Miniprep the GFP samples (Sunday afternoon)
4. Submit (GFP + AviTag + pZE12) for sequencing
5. Set up PCR reaction for VioB gene and repeat the ligation steps (next week)

Afternoon lab work done by: Youjin Cho, Charlie Chung

Objective

• Prepare a subculture of bacteria with (RFP + AviTagged pZE12) plasmid for production of RFP
• Transformation of (GFP + AviTagged pZE12 backbone) into DH5α electrocompetent bacteria and plated with 1:1000 ampicillin.
• Prepare freezer stocks of DH5α bacteria transformed with (RFP + AviTagged pZE12) and AviTagged pZE12
• Miniprep remaining volume of (RFP + AviTagged pZE12) and AviTagged pZE12 cultures

Preparing a Subculture

• Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Thursday evening

- Purpose of preliminary OD reading is to determine how much RFP sample you need to add to a new 25mL culture

- Use a 1:10 dilution of sample to minimize error when running the spectrophotometer

• Control: 1000µL LB
• RFP Sample: 900µL LB + 100µL (RFP + AviTagged pZE12) Colony #5

• RFP Sample OD = 0.283 (treat as [bacteria with RFP]), which translates to actual OD of 2.83 in Thursday's 5mL culture tube (after undoing the 1:10 dilution)

• Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture

(2.83)(? µL) = (desired beginning [RFP bacteria] = 0.05)(25mL = 25000µL)

? = 441.7µL Thursday's RFP bacteria culture to 25mL LB + 25µL ampicillin

• Incubate new 25mL RFP bacteria subculture in 37°C shaker for ~2 hours and 45 minutes
• At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG
• Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at 7:25pm)
• Incubate induced 25mL culture flask on room temperature shaker

Preparation of Freezer Stock

• 1:1 volume ratio of 30% glycerol and bacteria culture media

- Added 500µL 30% glycerol and 500µL (RFP + AviTagged pZE12) into three cryogenic storage tubes

- Added 500µL 30% glycerol and 500µL AviTagged pZE12 into three cryogenic storage tubes

- Stored in the -80°C freezer of Room 303

Miniprep

• Followed standard Qiagen Miniprep protocol
• NanoDrop spectrophotometry to determine DNA concentrations

AviTagged pZE12 backbone Sample 1 = 102.2ng/µL

AviTagged pZE12 backbone Sample 2 = 135.2ng/µL

(RFP + AviTagged pZE12 backbone) Sample 1 = 127ng/µL

(RFP + AviTagged pZE12 backbone) Sample 2 = 97.3ng/µL

Saturday, August 13

Afternoon lab work done by: Youjin Cho, Charlie Chung

Objective

• Lyse the 25mL culture of IPTG-induced (RFP + AviTagged backbone) bacteria
• Collect RFP protein

Procedure

1. Bring BugBuster Protein Extraction lysis buffer (EMD4Biosciences of Merck) to room temperature
2. Transfer 25mL culture to 50mL conical that is previously tared (later, we will need to measure mass of bacterial pellet). Centrifuge at 4500rpm for 25 minutes
3. Pour out supernatant. Measure mass of bacterial pellet. 0.4g * (5mL BugBuster per g of cells) = 2mL BugBuster
4. Rock for 20 minutes
5. Centrifuge at maximum rpm for 20 minutes at 4°C
6. Collect supernatant (RFP will be soluble here)

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Evening lab work done by: Charlie Chung

• Attempted to pick and culture colonies of (GFP + AviTagged backbone). However, couldn't find keys to Room 303. Thus, I was unable to pick and begin 3mL cultures
• Sorry for creating more work, but please complete this task Sunday morning