Newcastle University Wetlab/8 September 2008

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Newcastle University

GOLD MEDAL WINNER 2008

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Home >> Wet Lab >> Newcastle Wet Lab Journal

Wet lab work was carried out from 4 August to 19 September, Mondays to Fridays. Please click on a day to see the lab notebook.

August
MTWTFSS
        [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/1_August_2008&action=edit 1] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/2_August_2008&action=edit 2] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/3_August_2008&action=edit 3]
[http://2008.igem.org/Newcastle_University_Wetlab/4_August_2008 4] [http://2008.igem.org/Newcastle_University_Wetlab/5_August_2008 5] [http://2008.igem.org/Newcastle_University_Wetlab/6_August_2008 6] [http://2008.igem.org/Newcastle_University_Wetlab/7_August_2008 7] [http://2008.igem.org/Newcastle_University_Wetlab/8_August_2008 8] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/9_August_2008&action=edit 9] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/10_August_2008&action=edit 10]
[http://2008.igem.org/Newcastle_University_Wetlab/11_August_2008 11] [http://2008.igem.org/Newcastle_University_Wetlab/12_August_2008 12] [http://2008.igem.org/Newcastle_University_Wetlab/13_August_2008 13] [http://2008.igem.org/Newcastle_University_Wetlab/14_August_2008 14] [http://2008.igem.org/Newcastle_University_Wetlab/15_August_2008 15] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/16_August_2008&action=edit 16] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/17_August_2008&action=edit 17]
[http://2008.igem.org/Newcastle_University_Wetlab/18_August_2008 18] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/19_August_2008&action=edit 19] [http://2008.igem.org/Newcastle_University_Wetlab/20_August_2008 20] [http://2008.igem.org/Newcastle_University_Wetlab/21_August_2008 21] [http://2008.igem.org/Newcastle_University_Wetlab/22_August_2008 22] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/23_August_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/24_August_2008&action=edit 24]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/25_August_2008&action=edit 25] [http://2008.igem.org/Newcastle_University_Wetlab/26_August_2008 26] [http://2008.igem.org/Newcastle_University_Wetlab/27_August_2008 27] [http://2008.igem.org/Newcastle_University_Wetlab/28_August_2008 28] [http://2008.igem.org/Newcastle_University_Wetlab/29_August_2008 29] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/30_August_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/31_August_2008&action=edit 31]
September
MTWTFSS
[http://2008.igem.org/Newcastle_University_Wetlab/1_September_2008 1] [http://2008.igem.org/Newcastle_University_Wetlab/2_September_2008 2] [http://2008.igem.org/Newcastle_University_Wetlab/3_September_2008 3] [http://2008.igem.org/Newcastle_University_Wetlab/4_September_2008 4] [http://2008.igem.org/Newcastle_University_Wetlab/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/6_September_2008&action=edit 6] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/7_September_2008&action=edit 7]
[http://2008.igem.org/Newcastle_University_Wetlab/8_September_2008 8] [http://2008.igem.org/Newcastle_University_Wetlab/9_September_2008 9] [http://2008.igem.org/Newcastle_University_Wetlab/10_September_2008 10] [http://2008.igem.org/Newcastle_University_Wetlab/11_September_2008 11] [http://2008.igem.org/Newcastle_University_Wetlab/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/14_September_2008&action=edit 14]
[http://2008.igem.org/Newcastle_University_Wetlab/15_September_2008 15] [http://2008.igem.org/Newcastle_University_Wetlab/16_September_2008 16] [http://2008.igem.org/Newcastle_University_Wetlab/17_September_2008 17] [http://2008.igem.org/Newcastle_University_Wetlab/18_September_2008 18] [http://2008.igem.org/Newcastle_University_Wetlab/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/21_September_2008&action=edit 21]
[http://2008.igem.org/Newcastle_University_Wetlab/22_September_2008 22] [http://2008.igem.org/Newcastle_University_Wetlab/23_September_2008 23] [http://2008.igem.org/Newcastle_University_Wetlab/24_September_2008 24] [http://2008.igem.org/Newcastle_University_Wetlab/25_September_2008 25] [http://2008.igem.org/Newcastle_University_Wetlab/26_September_2008 26] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/28_September_2008&action=edit 28]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University_Wetlab/29_September_2008&action=edit 29] [http://2008.igem.org/Newcastle_University_Wetlab/30_September_2008 30]

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.

  • Overnight (ON) cultures were made by JW of iGEMgfp 7.1, iGEMgfp colonies 11.1, iGEMcherry 13.1 and 13.2.

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.

- 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.
  • Starch, sucrose and glucose agar plates were made (see protocol) and the following colonies streaked:

- 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

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

  • 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.

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.

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