Team:Newcastle/Labwork/16 October 2009

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Contents 1 Sending parts to the registry 1.1 Stochastic Switch 1.2 AND Gate 1.3 KinA 2 Microscopy Test to characterize KinA

Sending parts to the registry

Today, we prepared the synthesised bricks and placed them to the box ready to send to the parts registry.

Stochastic Switch

Suspended 5ug of the DNA in PMK backbone with 30ul of PCR water. (Final concentration:166ng/ul)

To send to the parts registry (final concentration:6.7 ng/ul in 20 ul)

0.8ul of DNA
19ul of water

AND Gate

Suspended 5ug of the DNA in pSB1AT3 backbone with 30ul of PCR water. (Final concentration:166ng/ul) To send to the parts registry (final concentration:6.7 ng/ul in 20 ul)

0.8ul of DNA
19ul of water

KinA

Measured the concentration of KinA as 64ng/ul Prepared the tube to send to the registry (6.4ng/ul)

 2ul Kina in PMK
 18ul of PCR water

Microscopy Test to characterize KinA

We labelled the five overnight cultures with 10ml of LB+ B. subtilis as below

1. Wild type B. subtilis (- Control)
2. B. subtilis transformed with pgdp-rrnb+KinA biobrick (+ Control)
3. BFS867, transformed with pgdp-rrnb+KinA biobrick
4. BFS840, transformed with pgdp-rrnb+KinA biobrick
5. BFS82426, transformed with pgdp-rrnb+KinA biobrick

We prepared the flasks as below

• Flask1(-):5ml overnight culture 1 + 60ml LB
• Flask1(+):5ml overnight culture 1 + 60ml LB
• Flask2(-):5ml overnight culture 2 + 60ml LB + CHL
• Flask2(+):5ml overnight culture 2 + 60ml LB + CHL
• Flask3(-):5ml overnight culture 3 + 60ml LB + CHL + Em
• Flask3(+):5ml overnight culture 3 + 60ml LB + CHL + Em
• Flask4(-):5ml overnight culture 4 + 60ml LB + CHL + Em
• Flask2(+):5ml overnight culture 4 + 60ml LB + CHL + Em
• Flask5(-):5ml overnight culture 5 + 60ml LB + CHL + Em
• Flask5(+):5ml overnight culture 6 + 60ml LB + CHL + Em

Placed the flasks in shaking inwater bath at 37C. After an hour later we added IPTG to the relevant flasks. Every half an hour we took samples from each flask.

WT- WT+ WT_rrnb- WT_rrnb+ BFS867- BFS867+
Time Point 1 0.174 0.163 0.183 0.184 0.161 0.154
Time Point 2 0.246 0.254 0.26 0.274 0.222 0.21
Time Point 3 0.524 0.487 0.497 0.502 0.339 0.319
Time Point 4 0.84 0.824 0.825 0.806 0.518 0.432
Time Point 5 1.065 1.081 0.974 1.022 0.992 0.851
Time Point 6 1.248 1.28 1.225 1.243 1.1311 1.214
Time Point 7 1.315 1.343 1.325 1.277 1.342 1.341