Melb:Adhesion

From 2007.igem.org

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1/2YFP-Fos & 1/2YFPJun (exists?parts: BBa_J40004 & 40006) Based on PsfA promotor (new) from Dr Alan Grossman (M.I.T.)

This part is based on the paper “Autotransporters as Scaffolds for Novel Bacterial Adhesins: Surface proteins of Esherishia Coli Cells displaying Jun/Fos Dimerization Domains” By Estaban Veiga, Víctor de Lorenzo, and Luis Angel Fernández.

This part was to be based on the split YFP from McGill team 1 2006 but it was unavailable. We reasoned that by having two Ecoli lines that express one of the McGill components each when stimulated by the photodetection system we can get the two to adhere. FOS-Beta-YFPC BBa_J40004 JUN-Beta-YFPN BBa_J40006

Many potential problems here:

  1. “complementation falls apart after a few hours at room temperature”
  2. “PST sites present in construct-not a biobrick”
  3. “not sure if complementation occurs when touching or simply close”
  4. “expression fragile wipped out overnight”
  5. “FOS part fluoresces without JUN part”
  6. “sometimes cluster, not always!!!!”
  7. “sometimes over whole single cell”

(1 to 3 see  : [[1]]) (4 to 7 see  : [[2]])

We originally considered a cadherin expressed on the surface but this opened up the question of how to get it there.

APPENDIX 1 : McGill method for processing Spanish bacteria from: http://2006.igem.org/wiki/index.php/More_Detailed

The vector plasmids Fos-Beta and Jun-Beta arrived from Spain on the 21st of June 2006. They arrived in the form of bacteria in agar tubes. This is from a Spanish group that bubplished the paper “Autotransporters as Scaffolds for Novel Bacterial Adhesins: Surface proteins of Esherishia Coli Cells displaying Jun/Fos Dimerization Domains” By Estaban Veiga, Víctor de Lorenzo, and Luis Angel Fernández. The plasmids had chloramphenicol resistance.

They have been seeded on June 28 at 11:45 am in 2ml 1X LB (without glucose) and 210ng of chloramphenicol (105ng/ml). This grew at 37OC until 20:00pm. At 20:00 pm 500μL of seedings were diluted to 25ml of 1X LB and 750ng chloramphenicol (30ng/ml). This grew overnight and at 9:56 am on June 29th a midiprep was performed using a quiagen midiprep kit. Each plasmid was dissolved in 50μL dH2O.

On July 4th the Jun-Beta plasmid midiprep DNA was confirmed by a restriction digest with BamHI that should have yielded two pieces at 1442bp and 3920bp. The digest was incubated at 37OC for 3 hours 23 minutes. This was ran on a 0.7% agarose gel with ethidium bromide and in comparison to known molecular weight markers, the fragments have been confirmed.

On July 6th the Fos-Beta DNA was confirmed by the exact same BamHI digestion at 37OC for 2h 20min, to yield pieces of similar size to the Jun-Beta, which it did. On July 28th the concentrations of the midiprep DNAs were found using spectrophotometry to be 5950ng/μL for Jun-Beta and 4500ng/μL for Fos-Beta. This was done using 1000 fold dilution of original stock, a spectrophotometer set at 250nm and Quartz cuvettes.

On August 16th both the midipreps were diluted 5 fold with dH2O.

On September 12th a double digest was performed on both vectors with SacII and EcoRI. The SacII is an enzyme that takes longer t digest than EcoRI. Therefore digestion was first made with SacII in NEB buffer 4 using 5μL of DNA. These digests had a total volume of 50μL. They were incubated at 37OC for 3 hours. After about 3 hours 1μL of the digest was ran on a 0.7% agarose gel with ethidium bromide and in parallel lanes each uncut plasmid (point of having these lanes is to see cut compared to uncut, supercoiling). This indeed confirmed that the SacII hadentirely cut he Jun and almost entirely for fos.

To the sacII digests, ecoRI was added along with ecoRI buffer and diluted up to 100μL in a digest consisting of a half half mix of ecoRI buffer and buffer 4. This was incubated at 37OC for 1h.

Then the enzymes were deactivated by heating at 65OC for 20 minutes. From the gel, by comparing to DNA ladder known marker DNA concentrations and using an optical system that quantitatively measures the intensity of the bands, the concentrations of the cut vectors were found to be 28.18ng/μL for Fos-Beta and 8.01ng/μL for Jun-Beta.