Team:TecMonterrey GDL/LabStrategies

Lab strategies

Overview

For our project, we aimed to develop a microorganism that could respond to changes in the concentration of extracellular glucose by generating a readily detectable signal. For this, we selected the membrane bound receptor Trz as a molecular sensing element. This is a fusion protein comprised of the periplasmic domain of the chemoreceptor Trg, which is fused to the intracellular domain of the osmosensor EnvZ. Both Trg and EnvZ proteins are endogenous receptors that bacteria use to sense biochemical changes in the environment, which are then transduced into the cell to influence gene expression, cell motility and behavior.

Due to the difficulties related to the use of custom knockout strains such as ΔompR-envZ, we selected a wild-type E. coli strain (NEB 5-alpha) as a chassis for our experiments. However, the presence of the endogenous EnvZ/OmpR system in wild-type bacteria could interfere with the signaling between the TRZ1 receptor and reporters under the control of the OmpR promoter (pOmpC). This is mainly because the activation of TRZ1 would lead to the phosphorylation of endogenous OmpR and thus, overexpression of genes under the control of the pOmpC. The resulting increase in the number of membrane porins could disrupt the osmoregulatory ability of the bacteria and hinder cell viability. Furthermore, transgenes coupled to the control of pOmpC could be involuntarily expressed by the activation of the endogenous EnvZ/OmpR system, which could lead to false positives.

Because of the interplay between TRZ1 signaling and pOmpC activation, we first aimed to independently characterize the influence of both genetic modifications in bacterial viability. First, we coupled Part:BBa_R0082 (pOmpC) to the expression of the fluorescent reporter GFP (Part:BBa_E0240). We then designed a new part comprised of the TRZ1 receptor (Part:BBa_K216004) under the control of the pBAD strong promoter (Part:BBa_K206000). We also designed a new part comprised of the TRZ1 receptor under the control of pOmpC to study the effect of this positive feedback loop in cell viability. This approach allowed us to evaluate the influence of the endogenous EnvZ/OmpR system in the glucose-sensing capabilities of our wild-type strain.

Hypothesis

The activation of the TRZ1 receptor and the subsequent stimulation of the endogenous EnvZ/OmpR system in a wild-type NEB-5 strain can be used to detect changes in the concentration of extracellular glucose through the activation of the pOmpC coupled to the synthesis of a readily detectable reporter.

Objective

Develop a glucose biosensor based on a genetically modified organism that can couple changes in the concentration of extracellular glucose to the generation of a readily detectable signal.

 Protocols

This are all the protocols we used during iGEM 2019. Here, a summary of each is provided, as well as the necessary materials and methods. If you want more information you can click on the link to see the complete protocol.Or feel free to contact us at igem.tecgdl@gmail.com

Agarose gel electrophoresis

Agarose gel electrophoresis

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Materials

• Agarose
• 1X TAE
• Ethidium bromide (stock concentration of 10 mg/mL)

Methods

1. Mix 0.6 g of agarose powder with 40 ml 1X TAE in a microwavable flask to get a final concentration of 1.5% (w/v).

Note: Do not mix with water and make sure to use the same buffer as the one in the gel box.

2. Microwave pulses of 30-45 sec until the agarose is completely dissolved.

Note: Do not over boil, it will change the agarose concentration.

3. Let agarose solution cool down to about 50°C, usually when you can keep your hand on the flask.


4. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/ml. EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.

Pro-Tip: Usually about 2-3 μl of lab stock solution per 100 ml gel.

Caution: EtBr is mutagen, wear gloves, eye protection and lab coat.

5. Pour slowly the agarose into a gel tray with the well comb in place to avoid bubbles. If bubbles are formed, can be pushed away with a pipette tip.


6. Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 minutes, until it has completely solidified.

Loading Samples and Running an Agarose Gel

1. Add loading buffer to each of your DNA samples.

Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.

2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill gel box with 1X TAE (or TBE) until the gel is covered.

Pro-Tip: Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has been run, just as you would if you had not added EtBr to the gel in the first place.

4. Carefully load a molecular weight ladder into the first lane of the gel.


5. Carefully load your samples into the additional wells of the gel.


6. Run the gel at 90V until the dieline is approximately 75-80% of the way down the gel during 1 hour.

Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.

7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.


8. Using any device that has UV light, visualize your DNA fragments.

Pro-Tip: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.

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Transformation by NEB 5-alpha Competent E. coli cells

Transformation by NEB 5-alpha Competent E. coli cells

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Description

There are many options when it comes to transformation. The one we used to introduce our plasmid in the cell is based on the chemical modification of the membrane. The process is known as transformation by ultra-competition, which is recommended by New England Biolabs.

Materials

• NEB 5-alpha Competent E. coli cells
• Ligation mixture
• 1.5 ml microcentrifuge tubes
• SOC media

Methods

1. Defrost competent cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.


2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.


3. Place the mixture on ice for 30 minutes.

Caution: ​Do not mix.

4. Heat shock at 42°C for 30 seconds.

Caution: ​Do not mix.

5. Add 950 µl of room temperature SOC into the tube.


6. Place tube at 37°C for 60 minutes. Shake at 250 rpm or rotate.


7. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.


8. Extend 50–100 µl of the cells and ligation mixture onto selection plates.

Note: ​Remember to previously warm selection plates to 37°C.

9. Incubate overnight at 37°C.

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Plasmid extraction by QIAprep® Spin Miniprep Kit

Plasmid extraction by QIAprep® Spin Miniprep Kit

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Description

The QIAprep Spin Miniprep Kit is designed for isolation of up to 20 μg high-purity plasmid or cosmid DNA for use in routine molecular biology applications, including fluorescent and radioactive sequencing and cloning.

Materials

• Bacterial overnight culture
• 2 ml conical microcentrifuge tubes
• 1.5 ml microcentrifuge tubes

The following materials were provided by Qiagen, QIAprep® Spin Miniprep Kit:

• Collection Tubes
• QIAprep 2.0 spin column
• Buffer P1 (2–8°C)
• Buffer P2
• Buffer N3
• Buffer PE
• Buffer EB (10 mM TrisCl, pH 8.5)

Methods

Before starting:

• Add the provided RNase A solution to Buffer P1, mix and store at 2-8°C.
• Add ethanol (96-100%) to the Buffer PE before use (see the bottle label for volume).
• All centrifugation steps are carried out at 13,000 rpm.

Preparation of lysate:

1. Pellet 2 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 minutes at room temperature (15-25°C).


2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.


3. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear.

Caution: Do not allow the lysis reaction to proceed for more than 5 minutes.

4. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.


5. Centrifuge for 10 minutes at 13,000 rpm in a microcentrifuge.


6. Apply 800 µl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting.


7. Centrifuge 30-60 sec and discard the flow-through.


8. Wash the QIAprep 2.0 spin column by adding 750 µl Buffer PE.


9. Centrifuge 30-60 sec and discard the flow-through.


10. Repeat step 9 to remove residual wash buffer.


11. Let 5 minutes drying the QIAprep 2.0 spin column to evaporate the rest of the residual wash buffer.


12. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube and 50µl Buffer EB to elute DNA. Let stand for 1 minute and centrifuge for 1 minute.


13. Quantify the concentration of DNA in Nanodrop.

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Digestion

Digestion

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Description

The digestion step is used to cut a specific target DNA sequence with specific restriction enzymes to obtain specific restriction sites (sticky ends in this case) for a subsequent ligation to a vector that will be used to transform an expression vector to express the desired protein.

Materials

• Target DNA
• 10X NEBuffer 2.1
• EcoRI-HF
• PstI
• Nuclease-free Water

Methods

1. Measure and set up reaction with the following, considering a 10 µl reaction:



COMPONENT

AMOUNT

Target DNA

100 ng

10X NEBuffer 2.1

1 µl (1X)

EcoRI-HF

0.1 µl (1 unit)

PstI

0.1 µl (1 unit)

Nuclease-free Water

Fill to 10 µl



2. Incubate at 37°C for 15 minutes.

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Ligation

Ligation

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Materials

• T4 DNA Ligase Buffer (10X)
• Vector DNA (2 kb)
• Insert DNA (1.7-2.1 kb)
• Nuclease-free water
• T4 DNA Ligase
• Microcentrifuge tubes

Methods

1. Setup the next 20 μl reaction in a microcentrifuge tube in ice with the following measures:

Pro-tip: We recommend to use NEBioCalculator to calculate molar ratios.

COMPONENT

AMOUNT

T4 DNA Ligase Buffer (10X)

2 μl

Vector DNA (2 kb)

0.040 pmol

Insert DNA (1.7-2.1 kb)

0.095-0.154 pmol

Nuclease-free water

Fill to 20 μ

T4 DNA Ligase

1 μl



2. Mix by pipetting the centrifuge up and down.


3. Incubate at 16°C overnight for 10 minutes.

Note: This step is necessary for cohesive/sticky ends. Skip this if blunt ends or single base overhangs wants to be achieved.

4. Alternatively, incubate at 16°C overnight for 2 hours.

Note: This step is necessary for blunt ends or single base overhangs.

Pro-tip: High concentration T4 DNA Ligase can be used in a 10 minute ligation.

5. Proceed to heat inactivation at 65°C for 10 minutes.


6. Transform 1-5 μl into 50 μl of competent cells after cooling down the reaction on ice.

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PCR

PCR

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Description

PCR (polymerase chain reaction) is a technique used to produce a great amount of copies of a DNA specific gene.

Material

• Template: cDNA, gDNA, λDNA
• Forward and reverse gene-specific primers
• Autoclaved, distilled water
• E-Gel® Gels, 1.2% (Cat. no. G-5018-01)
• TrackIt™ 1Kb Plus DNA Ladder (Cat. no. 10488-085)
• 0.2 or 0.5 mL nuclease-free microcentrifuge tubes

Methods

Note: The next procedure shows volumes for a 50 μL reaction.

1. Thaw, mix and centrifuge the reagents.


2. Next, set up the tubes on ice, and add autoclaved water, PCR SuperMix, 10 μM forward primer, 10 μM reverse primer and the template of DNA. Adjust the reaction volumes as needed. Cap the tube, mix and centrifuge.


3. Incubate the reaction in a thermal cycler, which repeats 25-35 the following cycle:
1. Initial denaturation. 94 for 2 minutes
2. Denature, 94℃ for 15 seconds
3. Anneal, 55℃ for 30 seconds
4. Extend, 72℃ for 1 minute/kb


4. Analyze 10 μL using agarose gel electrophoresis. When successful PCR occurs then can immediately use it, or store it at -20℃.

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SDS page

Notebook

This notebook provides a record of what we were doing at the laboratory during all this time and it was written at “Benchling”. The notebook only contains the name of the protocol and the date at we performed it.

Link to the Notebook

Notebook

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Contact Us:
igem.tecgdl@gmail.mx