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From 2008.igem.org

Newcastle University

GOLD MEDAL WINNER 2008

Home	Team	Original Aims	Software	Modelling	Proof of Concept Brick	Wet Lab	Conclusions

Lab Notebook

Meetings The team meets at least once a week on thursday afternoons. Details of each meeting can be found through the calendar links.

Ria and Jess finally started in the labs on Monday the 4th August. So we will start updating the wiki with what's been happening and the progress we are making.

Our construct (as seen above) contains:

• spaR (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene has an N-termianl domain that can be phosphorylated and a C-terminal domian that has DNA-binding properties ([http://http://aem.asm.org/cgi/reprint/59/1/296.pdf])
• spaK (subtilin peptide antibiotic Kinase) - the 387 amino acid peptide coded for by this gene phosphorylates spaR at its N-terminus ([http://http://aem.asm.org/cgi/reprint/59/1/296.pdf]). This activates the C-termainal DNA binding ability, and in turn positively regulates the expression of GFP by initiating its transcription

Aim 1: clone the spaRK system from pUC57 into pJWV021 and transform into DH5 alpha competent E. coli

Aim 2: clone the spaRK system from pUC57 into pGFP-rrnB and transform into DH5 alpha competent E. coli

The 2.2kb fragment (ncl08) contains the spaRK system and a promotor-less GFP gene linked to this. This means that when spaR is activated, its positive regulatory effect on spaK will in turn activate GFP.

Monday 4th August

All 5 of us (Megan, Mark, Nina, Ria and Jess) went into the lab today to decide on a plan of action for the weeks to come.

We used electrophoresis to check for the presence of plasmids (pGFPrrnB- our plasmid with the GFP gene, and pJWV021- with the mCherry gene). Our gel was inconclusive, so we decided to re-run it the following day to confirm our results.

In the afternoon we attempted to transform DH5 Alpha competent cells with DNA from the registry (from source plate 1010 well 4A and 1018 7A).

We followed the extraction method outlined in the green folder and for each DNA spot we plated two petri dishes: one with a larger volume of DNA (4μl) and one with a smaller volume (2μl) to make colony counting easier. We plated the following and incubated at 37˚C overnight:

Plate 1: +ve control (isolated plasmid plus TOP10 cells)

Plate 2: -ve control (TOP10 cells only, no plasmid)

Plate 3: Large 1010 4A

Plate 4: Small 1010 4A

Plate 5: Large 1018 7A

Plate 6: Small 1018 7A

Mark's Lab Journal

Nina's Lab Journal

May
M	T	W	T	F	S	S
			[http://2008.igem.org/Newcastle_University/1_May_2008 1]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/2_May_2008&action=edit 2]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/3_May_2008&action=edit 3]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/4_May_2008&action=edit 4]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/5_May_2008&action=edit 5]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/6_May_2008&action=edit 6]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/7_May_2008&action=edit 7]	[http://2008.igem.org/Newcastle_University/8_May_2008 8]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/9_May_2008&action=edit 9]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/10_May_2008&action=edit 10]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/11_May_2008&action=edit 11]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/12_May_2008&action=edit 12]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/13_May_2008&action=edit 13]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/14_May_2008&action=edit 14]	[http://2008.igem.org/Newcastle_University/15_May_2008 15]	[http://2008.igem.org/Newcastle_University/16_May_2008 16]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/17_May_2008&action=edit 17]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/18_May_2008&action=edit 18]
[http://2008.igem.org/Newcastle_University/19_May_2008 19]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/20_May_2008&action=edit 20]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/21_May_2008&action=edit 21]	[http://2008.igem.org/Newcastle_University/22_May_2008 22]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/23_May_2008&action=edit 23]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/24_May_2008&action=edit 24]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/25_May_2008&action=edit 25]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/26_May_2008&action=edit 26]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/27_May_2008&action=edit 27]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/28_May_2008&action=edit 28]	[http://2008.igem.org/Newcastle_University/29_May_2008 29]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/30_May_2008&action=edit 30]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/31_May_2008&action=edit 31]

June
M	T	W	T	F	S	S
						[http://2008.igem.org/wiki/index.php?title=Newcastle_University/1_June_2008&action=edit 1]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/2_June_2008&action=edit 2]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/3_June_2008&action=edit 3]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/4_June_2008&action=edit 4]	[http://2008.igem.org/Newcastle_University/5_June_2008 5]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/6_June_2008&action=edit 6]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/7_June_2008&action=edit 7]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/8_June_2008&action=edit 8]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/9_June_2008&action=edit 9]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/10_June_2008&action=edit 10]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/11_June_2008&action=edit 11]	[http://2008.igem.org/Newcastle_University/12_June_2008 12]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/13_June_2008&action=edit 13]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/14_June_2008&action=edit 14]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/15_June_2008&action=edit 15]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/16_June_2008&action=edit 16]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/17_June_2008&action=edit 17]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/18_June_2008&action=edit 18]	[http://2008.igem.org/Newcastle_University/19_June_2008 19]	[http://2008.igem.org/Newcastle_University/20_June_2008 20]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/21_June_2008&action=edit 21]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/22_June_2008&action=edit 22]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/23_June_2008&action=edit 23]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/24_June_2008&action=edit 24]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/25_June_2008&action=edit 25]	[http://2008.igem.org/Newcastle_University/26_June_2008 26]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/27_June_2008&action=edit 27]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/28_June_2008&action=edit 28]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/29_June_2008&action=edit 29]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/30_June_2008&action=edit 30]

July
M	T	W	T	F	S	S
	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/1_July_2008&action=edit 1]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/2_July_2008&action=edit 2]	[http://2008.igem.org/Newcastle_University/3_July_2008 3]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/4_July_2008&action=edit 4]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/5_July_2008&action=edit 5]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/6_July_2008&action=edit 6]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/7_July_2008&action=edit 7]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/8_July_2008&action=edit 8]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/9_July_2008&action=edit 9]	[http://2008.igem.org/Newcastle_University/10_July_2008 10]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/11_July_2008&action=edit 11]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/12_July_2008&action=edit 12]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/13_July_2008&action=edit 13]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/14_July_2008&action=edit 14]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/15_July_2008&action=edit 15]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/16_July_2008&action=edit 16]	[http://2008.igem.org/Newcastle_University/17_July_2008 17]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/18_July_2008&action=edit 18]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/19_July_2008&action=edit 19]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/20_July_2008&action=edit 20]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/21_July_2008&action=edit 21]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/22_July_2008&action=edit 22]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/23_July_2008&action=edit 23]	[http://2008.igem.org/Newcastle_University/24_July_2008 24]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/25_July_2008&action=edit 25]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/26_July_2008&action=edit 26]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/27_July_2008&action=edit 27]
[http://2008.igem.org/wiki/index.php?title=Newcastle_University/28_July_2008&action=edit 28]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/29_July_2008&action=edit 29]	[http://2008.igem.org/wiki/index.php?title=Newcastle_University/30_July_2008&action=edit 30]	[http://2008.igem.org/Newcastle_University/31_July_2008 31]

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