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From 2011.igem.org

August 14th - August 20th

Sunday, August 14

Afternoon lab work done by: Charlie Chung

• Picked three colonies from the two (GFP + Avi-Tagged pZE12 backbone) plates

- GFP + Avi + BB (1) #1, 2, 3

- GFP + Avi + BB (2) #1, 2, 3

• Incubating overnight in 37°C shaker of Room 304

• Re-picked three colonies from the (RFP + Avi-Tagged pZE12 backbone) plate in order to retry subculturing, induction, and cell lysis

- RFP + Avi + BB #7, 8, 9

• Incubating overnight in 37°C shaker of Room 304

Monday, August 15

Afternoon lab work done by: Youjin Cho, Maneesh Gupta

Objective

• Miniprep GFP samples that were cultured overnight and send them off for sequencing.
• Set-up the PCR for vioB using vio operon.

Miniprep & Sequencing

• Miniprepped the GFP samples using standard Qiagen Miniprep protocol.

GFP+Avi-Tag+pZE12 backbone (1)- 1,2,3

GFP+Avi-Tag+pZE12 backbone (2)- 1,2,3

• Set-up the samples for sequencing using the reverse primer.

Order number: 10254977

PCR Reaction

• Set-up the vioB PCR using the vio operon (= 20.3ng/μL).
• As Didi suggested, used the melting temperature of 53°C and annealing time for 3:30 minutes.

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Evening lab work done by: Maneesh Gupta, Charlie Chung

Preparing a Subculture

• Set up two cuvettes for preliminary optical density (OD) reading: (1) control (2) RFP sample from Sunday 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 + Avi-Tagged pZE12) Colony #9

• RFP Sample OD = 0.329 (treat as [bacteria with RFP]), which translates to actual OD of 3.29 in Sunday's 3mL culture tube (after undoing the 1:10 dilution)
• Use dilution equation to determine how much RFP bacteria culture is needed for the 25mL culture

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

? = 380µL Sunday'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 (6:30pm start)
• At end of incubation time, check OD. Target OD = 0.6-0.8, which means ready for induction of RFP production via IPTG

OD was 0.49 at 9pm. Let culture grow until 11pm for induction (IPTG addition).

• Induce 25mL RFP bacteria culture with 25µL 1M IPTG for desired 1mM addition (completed at 11pm)
• Incubate induced 25mL culture flask on room temperature shaker

Tuesday, August 16

Afternoon lab work done by: Maneesh Gupta, Charlie Chung

• Repeated cell lysis protocol (see Saturday, August 13) on 25mL culture from Monday.

- However, after centrifuging, the pellet was not red.

Troubleshooting Possibilities

• RFP being expressed in such little quantity that it cannot be confirmed by the naked eye

- Instead, RFP expression can be confirmed with a western blot on the cell lysate using an antibody for Avi-Tag

• Distance between the ribosomal binding site (RBS) and the AUG start codon of the RFP mRNA may be too long

- Redesign primers for the sake of cloning and expressing RFP

What We Did

- Picked a culture sample from the pellet formed after the centrifuge step

- Incubated in the 37°C shaker of Room 304 for Miniprep and sequencing tomorrow

p.s. -- Submit Didi's sequencing samples along with RFP sample!

Wednesday, August 17

Morning lab work done by: Youjin Cho, Charlie Chung

Miniprep DNA Plasmid Purification

• Miniprepped culture from bacterial cell pellet of (RFP + Avi-Tagged pZE12)

- Followed standard Qiagen Miniprep protocol

- NanoDrop spectrophotometry: 52.7ng/μL

Preparation for DNA Sequencing Submission

- 1μL reverse primer for pZE12

- 17μL Miniprep-purified (RFP + Avi-Tagged pZE12)

• Order Number: 10255141

Thursday, August 18

Friday, August 19

Lab work done by: Claire Paduano and Youjin Cho

Objective

• PCR clean up VioB insert
• Digest VioB insert and VioE in Avi-Tagged pZE12 vector backbone and gel purification
• GFP sequencing came back: unsuccessful. PCR off GFP overnight to try ligation again

Digestion Setups

• VioE in Avi-Tagged pZE12 vector backbone

19.05μL H2O

23.2μL VioE in Avi-Tagged pZE12 vector backbone (2μg)

5μL 10x NEBuffer 2

0.5μL 100x BSA

1.25μL KpnI

1μL HindIII

50μL Total

• VioB insert

38.25μL H2O

4μL VioB(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 0.25uL more KpnI to digestion mixture

Incubate in 37°C water bath for 2 hours

Gel purification of VioB insert and Avi-Tagged pZE12 vector backbone

• VioB 5.4 ng/uL
• Avi-Tagged backbone 11.4 ng/uL

Saturday, August 20

Morning lab work done by: Claire Paduano

Objective

• Set up ligation of VioB in Avi-Tagged backbone

Ligation

• VioB + Avi-Tagged Backbone

2.3μL Avi-Tagged pZE12 vector backbone

14.7μL VioB

0.0μL 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 at room temperature for 4hrs.

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Afternoon lab work done by: Youjin Cho, Charlie Chung

vioB Transformation

• Desalted the (vioB + Avi-Tag + BB) ligation reaction product and the (Avi-Tagged BB) control
• Transformed (vioB + Avi-Tag + BB) and the (Avi-Tagged BB) control into DH5α bacteria via electroporation
• Plated onto (LB + ampicillin) dishes and incubated overnight at 37°C

Alternative Method for RFP Expression

• Sequencing results from Wednesday's (August 17) sample show that the RFP gene was present in the Avi-Tagged pZE12 vector backbone
• Transformed 1.5μL of (RFP + Avi-Tag + BB) Miniprep-purified plasmid into DH5α bacteria with the IPTG gene via electroporation

- Ideal Results: Colonies that have grown tomorrow will be red because RFP-production is constitutively on

• Plated onto a (LB + ampicillin) dish and incubated overnight at 37°C