More Detailed

From 2006.igem.org

""More detailed:""

""The Vectors:""

• 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.

"""Inserts:"""

-PCR template:

• On the 27th of June, what was to be the PCR template for both our inserts arrived from Michigan in the form of DNA. The plasmid was called pDsBiFc- bJunYN155- bFosYC155. This plasmid has Amp resistaqnce.

• 3μL of this DNA has immediately been transformed by heat shock into Top 10 chemically competent cells (One Shot). This was plated on two Ampicillin plates, 150μL, and 50μL transformant.

• On June 28th, it was found that the plates had colonies.

• On July 6th, 10 seedings from plates were done by 13:10 (in 2ml LB and Amp). They were incubating until 22:00 pm (8 hours 50 minutes) when they were re-suspended in QUIAGEN buffer P1 (by spinning down, removal of supernatant, adding 250μL buffer P1), and placed in fridge.

• On July 10th, the re-suspended 9 of the seedings were re-seeded each in 5ml LB and Amp. They were placed at 37OC in shaker at 16:45pm.

• On July 11th, The seedings were incubating very long until 15:30 pm (22h 45min) before a standard QUIAGEN miniprep procedure was performed on all of them.

• On July 17th, first the miniprep DNA required purification using a QUIAGEN purification kit. Then, 8 double digests of purified DNA were se up at 18:35pm. They consisted of EcoRI, BamHI, NEB EcoRI buffer, and BSA. They were left at 37OC overnight.

• The digests were run on a 0.8% agarose gel with ethidium bromide. The result of the gel is that the first of the eight digests had the expected band location, one at 4000bp and the other at 800bp.

• To serve as a template, a 50X dilution of the first miniprep was performed.

PCR primers:

• Note: we are using two different sets of primers to PCR out two halves of YFP that are in the same plasmid pDsBiFc- bJunYN155- bFosYC155 (2 fusion proteins, 1 plasmid)

• On July 5th, the primers were designed, ordered and arrived. Note: The melting temperatures were determined using software called Primer Express. So four primers, both forward primers had EcoRI site in the forward primer and SacII site in the back Primer

• On July 7th, the dry primers were diluted with QUIAGEN buffer EB. (236μL EB Jun forward, 220μL EB Jun reverse, 170μL EB fos forward, 211μL EB Fos reverse.

-PCR:

• On July 27th, four PCR reactions were set up, two for each YFPN and YFPC (two halves of YFP). This was in a 100μL reaction vial each consisting of 80μL PCR dH2O, 10μL PCR buffer, 4μL dNTP’, 2μL each forward and reverse primer, 1μL BSA, 1μL Taq DNA Polymerase, 1μL Template (diluted pDsBiFc). Tese reaction tubes have undergone PCR. Following this the PCR solution was purified using PCR purification kit.

• 2.5μL of PCR products were run on a 1.2% agarose gel with ethidium bromide. On July 28th, using band comparison with ladder, the concentration of the PCR products was found to be about 25ng/μL (the intensity of all four bands was about the same).

-Preparation of insert by digestion:

• On August 23, a digestion of 20μL of the first of each PCR reactions was performed. EcoRI digestion was set up with NEB EcoRI buffer at 37OC for 3 hours. This was followed by running on a 1.2% agarose gel with ethidium bromide for about 15-30 minutes. Then a standard Gel extraction was performed using a Quiagen gel extraction kit (columns). This was followed by an overnight digestion with SacII in buffer 4 at 37OC. Then on August 24th, the digests were heat deactivated at 65OC for 20 minutes and 6μL of them was ran on a 1.2% agarose gel with EtBr and from this gel, the concentration of the inserts was found to be 14.6ng/μL for YFPC (Fos) and 8.95ng/μL for YFPN (Jun).

"""Ligations"""

• On September 13th, two ligations have been performed. One was a ligation of YFPC to Fos-Beta and the other a ligation of YFPN to Jun-Beta. It was decided, that both ligations were to include 100ng of insert and 100ng of vector. Knowing the concentrations of YFPC insert to be 14.6ng/μL, it was determined that 6.85μL would be used. Knowing Fos-beta concentration of 28.18ng/μL, 3.55μL would be used. In the other ligation, the concentrations were too small for 100ng of each to be used. Therefore, 59ng of each was used to keep the total volume under 20μl. Volume of YFPN was 6.61μL and Jun-Beta was 7.39μL. These ligations were left overnight in a room of about 18OC.

• On September 14th, a transformation of the ligation product into homemade chemically competent Top 10 cells was performed and plated on chloramphenicol plates.

• On September 15th, it was found colonies grew on the plates and there were not too many or too few.

• On September 21st, a protocol referred to as “cracking” was performed. Several colonies were picked from each ligation, they were placed in 20μL sterile dH2O, mixed and 1μL was placed on another plate with labelled grid. This was heated at 95OC for 5 minutes and then PCR could be performed directly on these, so several PCR reactions were performed.

• On September 22nd, five of the YFPN (Jun) PCR products were loaded onto a 1.2% gel. Bands of the right sizes were found (486bp) and that was the case for four out of the five wells had the right bands. On September 25th, nine of the YFPC (Fos) PCR products ran on a gel and all resulted in the correct band at 281bp.

• On September 26th, 2mL LB seedings with 2ng/ml chloramphenicol of colonies two of each ligation were performed and placed in 37OC Shaker. Later that day a standard miniprep procedure was ran and on September 27th, a transformation into commercial Bl21 cells has been done. On September 28th, two colonies of each transformant were seeded in 4mL LB overnight. On September 29th, iPTG induction began: diluted 500μL of each seeding into a 5ml LB and chloramphenicol seeding for each, as well as another dilution of 1mL into 5mL. This grew in 37OC shaker and after 1 hour the OD measurement (600nm) was 0.318 for Jun 1L and 0.248 of Fos 500μL. They were induced with the addition of 0.3μL iPTG. They were grown for 1 hour and were spun down, dissolved in water (LB is fluorescent) and viewed under the microscope. The result appeared that when added together, sells did clump together and glow yellow. Therefore, this was a success. However, unfortunately the induced cells did not last over the weekend in the fridge in LB.

• This success was repeated once more with another transformation. This time, on Wednesday October 25th, the iPTG induction was 3 hours and the cells were not spinned in centrifuge, instead they have undergone sedimentation at 1G. Some of them were mixed and incubated together while mixed. Again, a few hours later this induction did not last.