Team:Newcastle/Labwork/16 October 2009

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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.

We used the samples from time point 1 and 5 for the microscopy.

• We spinned the tubes for 2 min at 13000rpm
• Discarded the LB
• Resuspended in 500ul of SMM
• Spinned again and discarded SMM
• Resuspended in 50ul of SMM
• Prepared the samples for microscopy

Labellling the tubes for the microscopy

1. Tube 1, Time point 5, IPTG -
2. Tube 2, Time point 5, IPTG -
3. Tube 3, Time point 1, IPTG -
4. Tube 3, Time point 1, IPTG +
5. Tube 3, Time point 5, IPTG -
6. Tube 3, Time point 5, IPTG +
7. Tube 4, Time point 1, IPTG -
8. Tube 4, Time point 1, IPTG +
9. Tube 4, Time point 5, IPTG -
10. Tube 4, Time point 5, IPTG +
11. Tube 5, Time point 1, IPTG -
12. Tube 5, Time point 1, IPTG +
13. Tube 5, Time point 5, IPTG -
14. Tube 5, Time point 5, IPTG +

Pictures display the results from the 5th and 6th cultures. The 5th and 6th samples were taken from BFS667 transformed with pgfp-rrnb. The 5th one does nto have any IPTG hence no GFP whereas the 6th one has lots of GFP with sporulation. Hence we can conlude that sporulation was triggered by our biobrick. :) We managed to get the results we expected. Congrulations to everyone in the team!

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