Team:Newcastle University/Notebook

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Newcastle University

GOLD MEDAL WINNER 2008

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


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Introduction

Our construct:

Figure 1: Newcastle's iGEM team construct.

Fig 1 shows the construct which contains:

  • spaRK promotor
  • rrnB - rRNA binding site
  • spaR (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal 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) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of spaR [http://http://aem.asm.org/cgi/reprint/59/1/296.pdf]. This activates the DNA binding ability of the C-terminus of spaR, which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is gfp.
  • gfp (green fluorescence protein) - the marker being used to show activation of the spaRK system and therefore diagnosis of gram-positive bacteria by B. Subtilis
  • spaS promotor - a strong promotor inducible by upstream activation of spaRK. It can be placed in front any gene to regulate its activity.

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


Aim 1


Aim 2: clone the spaRK system from pUC57 into pGFP-rrnB and transform into TOP10 competent E. coli


Aim 2
  • The 2.2kb fragment (ncl08) contains the spaRK system and promotor-less gfp linked to this. This means that when spaR is activated, its positive regulatory effect on spaK will in turn activate gfp.

Aim 3: clone the agr system from the plasmid vector into pGFP-rrnB and transform into DH5 alpha competent E. coli


Aim 3
  • BBa_I647107 contains a partial agr operon, which includes agrC and A but has agrB and D deleted. This allows coding for the receptor (C/A) but not for production of the quorum peptides themselves (B/D). Eventually this will be linked to the spaRK system in a B. subtilis vector.

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Wet Lab Journal

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]