Team:Newcastle University/Notebook

From 2008.igem.org

Revision as of 22:59, 30 September 2008 by Mtaschuk (Talk | contribs)

Bugbuster-logo-red.png
Ncl uni logo.jpg


Newcastle University

GOLD MEDAL WINNER 2008

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


Home >> Wet Lab

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 [1]
  • spaK (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of spaR [2]. 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.

Wet Lab Journal

August
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
September
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30