Team:Tec-Monterrey/projectprotocols

1.Measure 60 ml of TBE (.5 X) with the probe.
2.Take a beaker and place 60 ml of TBE.
3.Place the filter paper on the scale.
4.Weigh 600mg of agarose gel on the scale.
5.Mix 600mg of agarose gel in TBE and heat it in the microwave for time intervals of 10 seconds.
6.Place the electrophoresis comb in the tray electrophoresis.
7.Pour the solution into the electrophoresis tray, making sure the level does not reach beyond the teeth of the comb. Wait until it solidifies.
8.Remove the electrophoresis comb and place the tray electrophoresis in the electrophoresis chamber.
9.Pour TBE by the sides of the electrophoresis chamber until the level of TBE exceeds the gel.
10.Mix the DNA samples, molecular weight ladder and DNA supercoil with 12μl of loading buffer and SYBR Green 4μl.
11.Load the wells with the samples made in the previous step.
12.Connect the electrophoresis chamber to the power supply at a voltage between 60V and 70V and run it for 1.5 hours.
13.Use transilluminator to see results.

1.Perform the necessary calculations using the calculator developed by the Tec-Monterrey 2010 team. (click here)
2.Add the required amounts of buffer, plasmid DNA, nuclease free water and BSA (keep reagents in ice except the sample DNA).
3.Pour the required amount of enzyme at the end.
4.Incubate for 1 hour at 37 ° C.

1.Take 1ml of transformed cells in a 1.5ml eppendorf tube.
2.Centrifuge for 1min at 10,000 rpm.
3.Discard the supernatant. Repeat step "1" 5 times.
4.Take 250μl of Cell Resuspension Solution to resuspend the pellet.
5.Take 250μl of Cell Lysis Solution. Immerse it 4 times and incubate for 4min.
6.Take 10μl of Alkaline Protease Solution and incubate for 4min.
7.Take 350μl of Neutralization Solution. Immerse it 4 times.
8.Centrifuge at 14,000 rpm for 10min.
9.Place the minicolumn in a collection tube.
10.Remove the supernatant with a point and place it in the minicolumn.
11.Centrifuge for 1min at 14,000 rpm.
12.Remove the liquid from the collection tube and replace the minicolumn.
13.Take 750μl of Column Wash Solution and place it in the minicolumn.
14.Centrifuge for 1min at 14,000 rpm.
15.Remove the liquid from the collection tube and replace the minicolumn.
16.Take 250μl of "Column Wash Solution" and place it in the minicolumn.
17.Centrifuge for 2 min at 14.000 rpm.
18.Remove the liquid from the collection tube. Take the minicolumn and place it in a 1.5ml microcentrifuge tube.
19.Take 100µl of nuclease free water and pour it in the minicolumn.
20.Centrifuge for 1min at 14,000 rpm.
21.Store at -20 ° C.

1.Cut the band of interest and weigh.
2.1μl of Mem Bind Solution will be placed for each mg of gel.
3.Heat the gel with a water bath at 50 ° C to dissolve the gel.
4.Incubate at 25 ° C for 1min.
5.Place the minicolumn with its collection tube.
6.Pour the liquid incubated in the minicolumn.
7.Centrifuge at 14,000 rpm for 2min.
8.Decant the solution of the collection tube and replace the minicolumn.
9.Add 750μl of Mem Wash Solution to the minicolumn.
10.Centrifuge at 14,000 rpm for 2min.
11.Decant the solution of the collection tube and replace the column.
12.Add 500μl of Mem Wash Solution to the minicolumn.
13.Centrifuge at 14,000 rpm for 2min.
14.Decant the solution of the collection tube and replace the minicolumn.
15.Centrifuge 1 min.
16.Take the mini-column and placed in a 1.5ml eppendorf tube.
17.Pour 35μl nuclease-free water in the minicolumn.
18.Centrifuge at 14,000 rpm for 1min.
19.Store at -20 ° C.

1.Cells should have an optical density of .4 (always keep tubes on ice).
2.Centrifuge at 5,000 rpm, 2 ° C for 5min.
3.Decant supernatant.
4.Add 1 ml of CaCl2 to resuspend the pellet.
5.Add 20ml CaCl2.
6.Centrifuge 5,000 rpm, 2 ° C for 5min.
7.Decant supernatant.
8.Add 1 ml of CaCl2 to resuspend the pellet.
9.Add 20ml CaCl2.
10.Centrifuge 5,000 rpm, 2 ° C for 5min.
11.Decant supernatant.
12.Add 500μl of glycerol + CaCl2.
13.Store at -80 ° C.

1.You need to have calcium competent cells.
2.Take 50μl of cells and mixed with 2μl of BioBrick resuspended.
3.Place on ice for 15min.
4.Place in microwave on level 2 for 1min.
5.Place on ice for 1min.
6.Add 200μl of LB.
7.Incubate for 1 hour at 37°C with stirring.
8.Cultivate in a dish 50µl of bacteria and 150µl in another dish.

1.Streak Escherichia coli Top10 cells onto LB-agar plate with no antibiotics and incubate at 37°C overnight.
2.Pick one colony and place it in a 50 mL tube with 20 mL LB medium. Incubate overnight on a shaker at 37°C and 350 rpm.
3.Add 250 ml of LB medium to a flask and add the overnight culture until an OD600 of 0.1 is reached.
4.Place the flask on a shaker at 37°C, 350 rpm until an OD600 between 0.4-0.6 is reached.
5.Transfer the diluted culture to 50 mL tubes.
6.After this step, the cells must be kept at 4°C at all times. Place the cells on ice for 15 minutes.
7.Cool the centrifuge to 4°C.
8.Centrifuge the tubes for 10 min at 8000g at 4°C.
9.Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
10.Centrifuge a second time for 10 min at 8000g at 4°C.
11.Remove supernatant and gently resuspend pellets with 10 mL cold sterile water by pipetting. Add the rest of the water to a total volume of 50 mL.
12.Centrifuge a third time for 10 min at 8000g rpm at 4°C.
13.Remove supernatant and gently resuspend pellets with the remaining water (if it’s too little, add some more).
14.Calculate and add glycerol so that the final concentration is 10-15 %.
15.Resuspend the cells and aliquot 50 μL per 0.2 mL tube (tubes on ice) and store at -80°C.

1.Chill electroporation cuvettes, DNA samples and tubes on ice.
2.Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice.
3.Turn on electroporator and set voltage to 2.5 kV.
4.Dial a micropipette to 1 or 2μL of DNA sample.
5.Dial a micropipette to 50μL of electrocompetent cells.
6.Dial a micropipette to 1000μL and pipet in SOC. Place micropipette on counter such that tip doesn't touch anything.
7.Pipet 1-2μL of DNA sample and place inside the cuvette.
8.Pipet 50μL of electrocompetent cells inside the cuvette ensuring they mix with the DNA sample. Do not pipet up and down.
9.Place cuvette back on ice to ensure it remains cold.
10.Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
11.Wipe off excess moisture from outside of cuvette.
12.Place in chamber of electroporator so that the cuvette sits between electrodes.
13.Pulse the cells with a shock by pressing button on electroporator.
14.Remove cuvette from the chamber and immediately add SOC.
15.Transfer cuvette to 37°C incubator and shake at 350 rpm to promote aeration. Incubate for 1 hr.
16.Plate 100 μL transformation onto LB-agar plate supplemented with appropriate antibiotic.
17.Incubate plate overnight at 37°C until colonies appear.

1.Take an Eppendorf tube and add the required amounts of reagents.
2.Mix gently and centrifuge so that the contents of the tube to the bottom.
3.Incubate at room temperature for 5 min.
4.Use 2μl of the ligation reaction to transform 100µl of competent cells.

1.In a microfuge tube mix in the following order:
a)10X Amplification Buffer (5µl)
b)20mM Solution of four dNTPs pH=8.0 (1µl)
c)20µM forward primer (2.5µl)
d)20µM reverse primer (2.5µl)
e)1-5 units/µl thermostable DNA polymerase
f)H2O (28-33µl)
g)Template DNA (5-10µl)
h)Total Volume (50µl)
2.Program the thermocycler at the necessary conditions for denaturation, annealing and polymerization.

This is the recipe to prepare an Acrylamide Gel (10%):

1. Place the resolving solution on the glass.
2. Add 1ml of water, why? Water help’s the gel to no have irregularities on the surface.
3. Let polymerize for 30 minutes.
4. Dry the water on the glass with filter paper.
5. Add the stacking solution.
6. Add the comb carefully, avoid the formation of bubbles.
7. Let stand for 1 hour and a half.
8. WATCH OUT acrylamide is neurotoxic, DO NOT swallow.
Note: Unlike other protocols, PSA cannot be prepared instantly. After you prepare it, you can refrigerate it at 4°C. When using PSA, It is highly recommended to avoid sudden temperature changes. You can use a cooler to prevent the temperature changes.
Note: PSA and TEMED are polymerizing agents (MUST NOT inhaled). Once both compounds are added, must be added nimbly to the solution in the glass, because these compounds may start polymerizing once it´s added. You need abilities, if you have a Thelma in your team leave her this work.

1.Place the preinoculum in wildtype and transformed cells in LB liquid medium.
2.Overnight at 35°C and 250 rpm.
3.Inoculate 6ml of LB liquid medium of each preinoculum.
4.Growth for 6 hours at 35°C and 250 rpm (OD = 0.6 - 1)
5.Induce with arabinose 1mM for several time at ypur specific conditions

1.6 ml of transformed cell culture is harvested by centrifugation at 12,000 x g for 1 min.
2.The supernatant is removed and the pellet is dried.
3.For each 1 g of cell pellet, 20 ml of xTractor Buffer, 40 µl of DNAse 200 µl of 100X lysozyme solution is added.
4.The suspension is incubated during 10 min at room temperature.
5.The crude lysate is centrifugated at 10 – 12000 x g for 20 min and the supernatant is called as soluble fraction and the pellet as insouble fraction.
6.The insoluble fraction is sonicated with water during about 5 sec.
7.50 µl of protein fraction is mixed with 50 µl of 2x sample buffer with 2-ME and heat 10 min at 95 °C.

1.Add 1.0 ml 0.05 M Na-citrate, pH 4.8 to a test tube of 50 ml volume.
2.Add 500 micro liters of citrate buffer.
3.Make 4 dilutions:
4.500 micro liters of citrate buffer + 1.5 mL enzyme
5.500 micro liters of citrate buffer + 500 micro liters of enzyme
6.500 micro liters of citrate buffer + 500 micro liters of enzyme
7.500 micro liters of citrate buffer + 500 micro liters of enzyme
8.Temperate to 50ºC , ad done filter paper strip, mix
9.Incubate 50ºC, 60 min.
10.Add 3.0 mL DNS, mix
11.Boil for exactly 5.0 min. In a vigorously boiling water bath containing sufficient water. After boiling, transfer to a cold water bath
12.Add deionized water to reach 20 mL of volume. Miz by completely inverting the tube several times so that the solution separates from the bottom of the tube at each immersion.
13.When the pulp has settled well, after at least 20 min, the color formed is measured against the spectro zero at 540 nm.
14.Spectro Zero: 1.5 mL citrate buffer + 3.0 mL DNS
15.Enzyme blank : 1.0 mL citrate buffer + 0.5 mL enzyme + 3.0 mL DNS

A.Preparation of the reagent DNS
1.Heat 300 ml of distilled water in a 1 L glass to a temperature of 50° C and dissolve 5 g of dinitrosalicylic acid. Shake it.
2.Add 50 ml of a 4 M NaOH solution (NaOH 8 take it to 50 ml).
3.Add 150 g of sodium and potassium tartrate and continue stirring and heating until the complete dissolution of solids.
4.Cool the dissolution to ambient temperature.
5.Transfer this solution to a flask of 500 ml and fill with distilled water.
6.The reagent will be stored in amber bottle at room temperature, avoiding that it be placed together with oxidizing or reducing agents and flammable substances.
7.The amber bottle labeled under the name of “DNS Reagent” and records the name of the project, the person responsible for the reagent and the date.
B.Calibration curve
1.Prepare 5 standard solutions of glucose with the following concentrations:
a.2.0 mM
b.1.5 mM
c.1.0 mM
d.0.5 mM
e.0.25 mM
2.The solutions prepared in point 1 will be used to perform the 5 corresponding points on the calibration curve.
3.Prepare 150 mL of stock solution of glucose 2 mM from solid D (+) - glucose
•100 ml flask requires 0.036 g of solid.
•50 ml flask requires 0.018 g of solid.
4.Separate 25 ml of this solution in small amber bottle and pour the rest into a 150 ml beaker for later use in dilutions.
5.A sample of the stock is transferred to the appropriate flask, it’s completed with distilled water, and finally, stored in a small amber vial of 25 ml properly labeled. The quantities are summarized in the following table:

* Note: to measure the milliliters of stock solution employ a 10 ml pipette and to measure the tenths employ a 1000 µl micropipette.
Note: The calibration curve should be tripled and the values to correlate (absorbance vs. standard concentration) should be the average of the replications.
A.Determination of reducing sugars in the sample.
1.Take 3 ml of each solutions of the calibration curve and transfer them into 15 ml tubes. Add 1 ml of the reagent DNS.
2.Additionally, prepare a blank solution. For blank, take 3 ml of distilled water and add 1 ml of reagent DNS.
3.The tubes are immersed boiling water for 5 minutes. After this time are dipped in cold water at room temperature.
4.Determine the absorbance of the sample @ 540 nm.

Sac C gene from Zymomonas mobilis was PCR amplified with the primers S1PSF: 5’-GAATT CGCGG CCGCT TCTAG AGGAG CTCAT GTTTA ATTTT AATGC CAGTC GC-3’, S1PSR 5’-CTGCA GCGGC CGCTA CTAGT AGCTA GCGTA TTTGC GACGA TCAGG G-3’. The amplified fragment was cloned in pGEM T Easy Vector. The amplification mixture for 50 mL contained 1U of Platinum Taq HF polymerase (Invitrogen), 60 mM Tris-SO₄, 18 mM Ammonium Sulfate, 0.2 mM for each dNTP, 2 mM MgSO₄, ¿? mM of forward and reverse primers. PCR was performed in an iCycler (BioRad) using the following program: 94 C for 3 min, 30 cycles of 94 C for 30 s, 56.4 C for 45 s, and 72 8C for 1 min, and finally an extension step at 72 C for 8 min. The PCR product cloned in pGEM T Easy Vector was added to the registry (BBa_K633003) and the product was cloned in the plasmid pSB1C3 and added to the registry (BBa_NEEEW).

The ompA+sacC construction was generated by joining the biobricks of a promoter (araC BBa_I13458 and PBAD BBa_K206000), RBS (BBa_), lpp+ompA (BBa_K103006) with the biobrick standard assembly protocol (cita). The expected DNA fragment size (xx bp) of the ompA + sacC construct was confirmed by several restriction endonuclease reactions and agarose gel electrophoresis, and used to transform the E.coli strins BL21SI, Rosetta Gami, XL1 Blue, C43 and BW27783.

The E. coli strains containing the ompA+sacC construct and non-trasnsformed strains as negative controls were cultured with 6 mL of media M9 with glycerol as carbon source at an initial optical density at 600 nm (OD600) of 0.1. The batch cultures were performed at 37C until the OD600 of 0.6 was attained. Then, the expression was induced with L-arabinose and the temperature of postinduction was changed to 15C. Culture samples collected from the bioreactor were harvested by centrifugation. The half was used for the whole cell assay and the other half was processed with Clontech x-Tractor kit to obtain soluble and insoluble fraction of each strain. Both fractions were separated by 12% SDS-PAGE and visualized with XXX% (w/v) Coomassie Brilliant Blue R250?