"

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.

---

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 VioE in Avi-Tagged 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 0.25uL 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
• avitagged backbone 11.4 ng/uL

Saturday, August 20