IGEM:MIT/2006/Notebook/2006-6-21

Results from yesterday's liquid culture experiment

200 uM and 300 uM concentrations of Salicylic Acid in both types of cells smelled the best. The minimal media seemed to have a weaker smell.

The Heat-Sensitive Promoters are in the Registry

hybB (cold shock promoter):

<bbpart>BBa_J45503</bbpart>

htpG (heat shock promoter):

<bbpart>BBa_J45504</bbpart>

SAMT Sequencing Results

1. All of them failed- there must be issue with SAMT reverse primer

SA Optimal Concentration

1. No carbon source for the plates so no cell growth
2. SA at 300 μM is best from liquid culture

New Smelling Experiment

Cells with SAMT and BSMT were grown up with varying concentrations of Salicylic Acid in the media. The following tubes were made with 20 uL of the appropriate cells and 7 mL of the appropriate media: [note: all quantities in uM are really ug/mL]

SAMT in LB Kan with 17.36 uL SA (200 uM)

SAMT in LB Kan with 21.70 uL SA (250 uM)

SAMT in LB Kan with 26.04 uL SA (300 uM)

SAMT in LB Kan with 30.38 uL SA (350 uM)

SAMT in LB Kan with 34.72 uL SA (400 uM)

BSMT in LB Kan with 17.36 uL SA (200 uM)

BSMT in LB Kan with 21.70 uL SA (250 uM)

BSMT in LB Kan with 26.04 uL SA (300 uM)

BSMT in LB Kan with 30.38 uL SA (350 uM)

BSMT in LB Kan with 34.72 uL SA (400 uM)

SAMT in -TRP with 26.04 uL SA (300 uM)

BSMT in -TRP with 26.04 uL SA (300 uM)

Controls:

SAMT in LB Kan with 26.04 uL SA (300 uM) (Not Induced)

BSMT in LB Kan with 26.04 uL SA (300 uM) (Not Induced)

To Do

1. ligate the cut BSMT into a biobrick backbone
   • use red Kan backbone
   • ligation reaction temperature is a compromise between competing reactions -- room temperature is pretty effective and fast (15 minutes for transformants)
     • note: T4 is optimal at 37c, but cannot run reaction at this temp b/c will get melting at this high of a temp. (sticky ends only have 8 H-bonds)
     • note: could alternatively try 18c/16c in fridge overnight
   • be kind to T4 buffer, can be less kind to T4 enzyme
   • want equal moles of insert and backbone (50 nanograms each)
   • run in smallest volume possible
2. run ATF1 pcr products on gel to see if pcr was successful at higher temperature
3. repeat BAMT and SAMT PCR at a higher temperature gradient
   • try 48-64 this time and place most tubes in middle region

indole

1. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium
   • http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14569285&dopt=Abstract
   • could we try to get this mutant e. coli 3714 strain?
     • if you can find the strain here: http://cgsc.biology.yale.edu/cgsc.html we can easily get it.
2. Here are 2 more articles on the mutant indole-negative e. coli strain 3714
   • I hope that we can get mutant strain 3714 -- (or make our own mutant) -- what do you guys think about e. coli biofilm and cell-cell signaling systems/applications?? anyways, take a look...
   • 1. Martino PD, Fursy R, Bret L, Sundararaju B, and Phillips RS. Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria. Can J Microbiol. 2003 Jul;49(7):443-9. DOI:10.1139/w03-056 |

1. • 1. Di Martino P, Merieau A, Phillips R, Orange N, and Hulen C. Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase. Can J Microbiol. 2002 Feb;48(2):132-7. DOI:10.1139/w02-001 |

Ligation reaction

1. dilute BSMT by adding 40 μL water
   • final concentration is 15ng/μL
   • add 1 μL + 1.5 μL to ligation
2. Ligation mix test (10 μL)
   • 1 μL T4 ligation buffer
   • .5 μL T4 enzyme
   • 6 μL backbone (kan) (40 nanograms)
   • 1 μL diluted BSMT
   • 1.5 μL water (15 nanograms)
3. Ligation control test (10 μL)
   • 1 μL T4 ligation buffer
   • .5 μL T4 enzyme
   • 6 μL backbone (kan) (40 nanograms)
   • 2.5 μL water

ATF1 gel results

1. Unsuccessful at higher temperatures (48-64)
   • need to reorder new (longer) primers

Made double antibiotic resistant plates

• 20 Kan/Amp plates: 1mL Kan, .5mL Amp in 500mL of LB/agar [GREEN for Kan, ORANGE for Amp]
• 20 Kan/Cam plates: .5mL CHL, .5mL Amp in 500mL of LB/agar [BLUE for CHL, ORANGE for Amp]
• TOM'S PLATES: ORANGE = AMP, GREEN = CHL

Set up BAMT & SAMT PCR with Tom's new PCR mix

• General formula for one PCR tube: 49μL of Tom's PCR mix + 1μL of appropriate template + .6μL of each primer