Newcastle University Wetlab/8 September 2008

Monday 08th September

 * Results from the agar cultures:

- iGEMgfp (chloramphenicol) colony 7 - many colonies

- iGEMgfp (chloramphenicol) colony 11 - many colonies

- iGEMgfp (chloramphenicol) negative control - no colonies

- iGEMcherry (kanomycin) colony 13 - a few colonies

- iGEMcherry (kanomycin) colony 14 - 3 colonies (plus 'background' contaminants)

- iGEMcherry (kanomycin) negative control - no colonies (some 'background' contaminants)


 * Agar stabs were taken from the following colonies and streaked onto agar to obtain isolated colonies:

- iGEMgfp colonies 7.1, 7.2, 7.3, 7.4, 7.5, 7.6

- iGEMgfp colonies 11.1, 11.2, 11.3, 11.4, 11.5, 11.6

- iGEMcherry colonies 13.1, 13.2, 13.3

B. subtilis wild type strain ATCC6633 was also streaked onto agar.

The following cultures were then made and incubated at 37˚C whilst shaking for 3 hours. Dilute (dil) cultures were made by adding 500μl ON culture to 10mL LB. After 3 hours ATCC6633 supernatent was obtained by centrifuging ON ATCC6633 culture and 250μl of this added to the + ATCC6633 tubes.
 * Overnight (ON) cultures were made by JW of iGEMgfp 7.1, iGEMgfp colonies 11.1, iGEMcherry 13.1 and 13.2.

- iGEMgfp ON + ATCC6633

- iGEMgfp ON - ATCC6633

- iGEMgfp dil + ATCC6633

- iGEMgfp dil - ATCC6633

- iGEMcherry ON + ATCC6633

- iGEMcherry ON - ATCC6633

- iGEMcherry dil + ATCC6633

- iGEMcherry dil - ATCC6633


 * These were left to incubate at 37˚C for a further 2 hours to allow gfp and mCherry induction by the subtilin contained in the ATCC6633 supernatent. Those wiothout supernatent were positive controls.


 * The B. subtilis cells were prepared for microscopy (see protocol) and viewed:

- iGEMgfp ON + ATCC6633 - some cells expressed gfp

- iGEMgfp ON - ATCC6633 - some cells expressed gfp (although less than in the presence of subtilin)

- iGEMgfp dil + ATCC6633 - some cells expressed gfp

- iGEMgfp dil - ATCC6633 - some cells expressed gfp (although less than in the presence of subtilin)

- iGEMcherry ON + ATCC6633 - very few celled expressed mCherry

- iGEMcherry ON - ATCC6633 - very few celled expressed mCherry

- iGEMcherry dil + ATCC6633 - very few celled expressed mCherry

- iGEMcherry dil - ATCC6633 - very few celled expressed mCherry


 * There appeared to be a difference in the GFP intensities between iGEMgfp with and without subtilin but this microscopy was not sufficient to confirm this.

- iGEMgfp 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6 streaked onto starch agar
 * Starch, sucrose and glucose agar plates were made (see protocol) and the following colonies streaked:

- iGEMcherry 13.1, 13.2, 13.3 streaked onto both glucose and sucrose

iGEMcherry should not be able to grow on sucrose as the insert should have integrated into sacA. The bacterium therefore cannot make sucrase to digest sucrose and consequently will have no carbon source. The glucose plates act as controls.
 * iGEMgfp should be able to grow on starch but will have no 'halo' surrounding the colonies (the halo is digested starch). This is becuase the insert should have integrated into amyE and the bacteria therefore cannot make amylase to digest starch.


 * Overnight cultures were also made of iGEMgfp 7.1 and 11.1 and iGEMcherry 13.1 and 13.2.