Week 1 – Expressing the SSB protein - PowerPoint Presentation

Slide1

Week 1 – Expressing the SSB protein

The SSB gene has been cloned (engineered) into a bacterial overexpression plasmid called pET21a. The SSB gene expression is controlled by the T7 promoter. To activate transcription from the T7 promoter, cells carrying the pET21a-SSB plasmid need to express the RNA polymerase from a bacteriophage origin. The name of this phase is T7. BL21-DE3 cells are specially engineered bacterial cells that carry the gene for the T7 RNA polymerase engineered into their chromosome. The T7 RNA polymerase gene in these cells is under the control of an inducible lac promoter. Therefore to activate expression of the SSB gene, the pET21a-SSB plasmid has to be transformed into the BL21-DE3 cells.

Step 1:

Transform BL21-DE3 cells with pET21a-SSB plasmid

BL21-DE3 cells that take up the plasmid DNA will form colonies on LB-Amp plates

Step 2:

Grow BL21-DE3 colony in LB-Ampicillin media and induce SSB expression by adding IPTG

Step 3:

Lyse cells and check expression using SDS-PAGE

Protein size marker

Gel Lanes

100kDa

50kDa

25kDa

No IPTG

With IPTG

SSB protein

Expression and purification of SSB protein from pathogenic bacteria

pET21a

Amp

r

Sofia Origanti and Edwin Antony - 2015

The T7 RNA polymerase gene in the BL21-DE3 cells and the T7 promoter in the pET21 plasmid are under the control of the lac-inducible system.

Lac operon-overview:

The lac operon involves the expression of three

lac

genes essential for lactose metabolism as a single transcript transcribed from a single promoter. It is an inducible system wherein the presence of lactose induces the expression of the

lac

genes.

In the absence of lactose, the expression of the

lac

genes is shut-off by the binding of the lac repressor protein to an operator sequence adjacent to the lac promoter. The lac repressor prevents the RNA polymerase from transcribing the

lac

genes.

When lactose is present, lactose binds to the lac repressor and inactivates it. Inactive repressor cannot bind to the operator. This allows RNA polymerase to transcribe the

lac

genes.

lacI

repressor

gene

lacZ

lacY

lacA

lac genesoperator

polymerasepromoterLac repressor binds to the lac operator and prevents RNA polymerase from transcribing the lac genes.

No transcriptionIn the absence of lactose :

lacI

repressor

gene

lacZ

lacY

lacA

lac

genesoperator

Lactose binds to the lac repressor and inactivates it. This prevents the lac repressor from binding to the operator. RNA polymerase is now free to transcribe the lac genesTranscription activated

In the presence of lactose :lactoseLac repressor

protein

The Lac operon

polymeraseLac repressor protein X

X

Sofia Origanti and Edwin Antony - 2015

Taking advantage of the lac-inducible system, the lac-promoter driven T7 RNA polymerase and the T7 promoter are engineered to be controlled by the addition of a stable lactose synthetic analog – IPTG (

Isopropyl β-D-1-thiogalactopyranoside )

Genomic DNA

T7 polymerase gene

lacI

– repressor gene

Lac

promoter

operator

promoter

pET21a

plasmid

Amp resistance gene

T7

Ori

operator

lacI

– repressor gene

SSB gene

T7 polymerase

protein

T7 polymerase binds to T7 promoter

IPTG

lac

repressor protein

IPTG inactivates lac-repressor

BL21-DE3 cell

In the absence of IPTG (lactose analog), transcription from the lac promoter and any leaky transcription from the T7 promoter is shut-off by the binding of the lac repressor protein to the operator sequence adjacent to the lac and T7 promoter.

When IPTG is added, it binds to the lac repressor and inactivates it. This activates transcription of the T7 RNA polymerase. T7 RNA polymerase binds to the T7 promoter. Since the T7 promoter is no longer inhibited by the lac repressor in the presence of IPTG, SSB expression is induced (in the presence of IPTG). SSB gene is transcribed to generate mRNA, which is subsequently translated to generate SSB protein.

Control of the Lac operon by IPTG

X

Sofia Origanti and Edwin Antony - 2015

Cell transformationThaw BL21-DE3 cells only on ice. Label a 1.5mL microcentrifuge tube with your gene name.Add 50 uL of BL21-DE3 cells to the tube.To the same tube, add 2 uL

of pET21a-SSB DNA.

Mix gently by tapping with fingers.

Incubate on ice for 5 min.

Heat shock by incubating tube at 42°C for 60 seconds.

Place the tube back on ice for 2 minutes.

Add 250 uL LB-media. Mix gently by tapping

To help cells recover from the heat shock, place the tube in an incubator at 37°C, and shake at 220rpm for an hour.

Next to a flame (careful!), plate the entire reaction on a LB-Ampicillin plate. Label with your group’s name and incubate the plate at 37°C overnight

Week 1. Protocols

Sofia Origanti and Edwin Antony - 2015

Step 2 – Overnight Starter Culture

Prepare a 15-ml culture tube with 5 ml LB media containing Ampicillin (50

ug/ml final concentration).Pick a colony from the plates transformed previous day (step 1) and grow overnight to generate a starter culture. Overnight incubation at 37°C with shaking at 220 rpm.

Induction of SSB production with IPTGAdd 2.5 mL of starter culture to 1 L of LB-growth media in a 2.8 L fernbach flask. Label with your groups’ name and incubate the flask at 37°C, shaking at 220 rpm. (TA will start this culture)After ~3 hrs, transfer 1ml of the culture into a cuvette and check if the cells have reached an optical density (OD) of 0.6 using a spectrophotometer set at 600 nm wavelengthWhen cells reach OD600

0.6, collect 1 ml of the culture in a 1.7mL micro centrifuge tube. Label with groups’ name and as “No IPTG” . Spin this culture at 13000 rpm in a table top centrifuge, discard the supernatant and freeze the cell pellet at -20°C.

To the large flask, add 400 ul of a 1 M IPTG stock (0.4 mM final) and continue shaking at 37°C at 220 rpm for 3

hrs (alternatively, this culture can be grown overnight at 20°C). This step is called “

induction of protein expression”.

Remove the flask after 3

hrs

of induction and transfer the cell culture solution into a 1000 mL centrifuge bottle. Please label the centrifuge bottle with your groups’ name. Also, collect 1 ml of the induced culture sample in a 1.7mL micro centrifuge tube. Label with groups’ name and as “with IPTG” . Spin this culture at 13000 rpm in a table top centrifuge, discard the supernatant and freeze the cell pellet at -20°C.

Collect cells from the large culture by spinning in a centrifuge at 4200 rpm for 15 min at 4°C. Decant the solution and save the pellets. Resuspend the cells with 20 ml of 1X PBS (phosphate buffered saline) and transfer to one 40 ml Falcon-tube. Spin the cells at 4200 for 10 min, decant the solution, and save the cell pellet at -80°C.

Resuspend the “ No IPTG” and “with IPTG” cells in 150 ul MQH

2

O by pipetting. Add 150 uL

of 2X SDS protein-loading dye to the tubes and boil in a heat block for 10 min. Spin the tubes for 1 min at 13000 rpm in a table top centrifuge and load 10 uL

of the samples on an 12 % SDS-PAGE gel. Please see the attached pdf file for background on SDS-PAGE and gel preparation protocol.

Week 1. ProtocolsSofia Origanti and Edwin Antony - 2015

Step 4: Checking protein expression using SDS-PAGE protocol

Prepare the SDS-gel using the following reagents:12% Resolving gel layer (8 mL total): DD water – 3.47 mL 40% acrylamide – 2.4 mL 1.5 M Tris pH 8.8 – 2.0 mL 20% SDS – 40 uL 10% APS – 80 uL *TEMED – 7.5 uL(mix everything and add Temed last –right before pouring the gel)4% Stacking gel layer (7.5 mL total): DD water – 5.8 mL

40% acrylamide – 0.8 mL 1 M Tris pH 6.8 – 940 uL

20% SDS – 40 uL 10% APS – 80 uL

*TEMED – 7.5 uL(mix everything and add Temed last –right before pouring the stacking gel layer)

Gel preparation:Clean the 1.5 mm glass plate and spacer plate with DD water and ethanol and wipe clean.

Assemble the gel plates using the Bio-Rad gel assembly cassette.

Pour 7.5 ml of the resolving gel layer. Immediately top the gel with isopropanol to ensure an even gel-loading layer.

Wait for 15 min for the gel to polymerize. Prepare the stacking Gel.

Decant the isopropanol and wash with dd water. Wipe down the assembly.

Pour the stacking gel till it reaches the top of the gel assembly. Slowly insert a clean 1.5 mm comb (inserting the comb at a slight angle-helps!)

Wait 15 min for the gel to polymerize and you can proceed to sample loading

Loading samples and running the gel:Disassemble the gel assembly and keep the gel plates with the comb intact.

Assemble the gel plates with the comb using the Bio-Rad gel loading cassette.

Add 1L of 1X running buffer (100 mL of 10X running buffer + 900 mL ddwater). Load 10uL of sample in SDS-loading dye. Load protein size ladder. Run the gel at 120 milli amps for 1 hr.

Week 1. ProtocolsSofia Origanti and Edwin Antony - 2015

Wear gloves and a lab coat during staining and

destaining. WARNING: the stain is very hard to remove from clothing. The solutions have methanol and acetic acid – hence do not touch them with your bare hands.Dismantle the gel rig, wash all the components with water and dry them. Wash the gel cassette gently under water to remove residual SDS buffer. Disassemble the gel cassette and gently place the gel into a plastic or glass dish. Tupperware is provided. Label the bottom container with your groups’ name and SSB strain. Date it as well.Add Coomassie stain solution. Make sure the entire gel is covered by the staining solution.

Place the cover, but do not close it completely. Microwave on high for 20 sec. Carefully remove the

tupperware from the microwave, fully close the lid, and then place on the rocker for 15 min.

Decant the stain in to the waste bottle (DO NOT pour the stain down the drain). Gently hold the gel with your finger and rinse it three times with tap water.

Add sufficient

destaining

solution to cover the gel.

Crumple a Kim wipe or paper towel and place it next to the gel inside the Tupperware. This soaks up the excess dye as it comes off the gel.

Cover the Tupperware and place on rocker for 1hr.

Analyze gel bands using a light box.

TA will transfer the gel to water overnight and you can image the gel next week.

Staining and

destaining

of SDS-PAGE

Week 1. ProtocolsSofia Origanti and Edwin Antony - 2015

Adapted from QED Bioscience

Week 1. Notes – Principles of SDS-PAGEHow SDS-PAGE worksSDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight. It’s one of those techniques that is commonly used but not frequently fully understood. So let’s try and fix that. SDS-PAGE separates proteins according to their molecular weight, based on their differential rates of migration through a sieving matrix (a gel) under the influence of an applied electrical field.Making the Rate of Protein Migration Proportional to Molecular WeightThe movement of any charged species through an electric field is determined by its net charge, its molecular radius and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge nor their molecular radius is molecular weight dependent. Instead, their net charge is determined by amino acid composition i.e. the sum of the positive and negative amino acids in the protein and molecular radius by the protein’s tertiary structure. So in their native state, different proteins with the same molecular weight would migrate at different speeds in an electrical field depending on their charge and 3D shape. To separate proteins in an electrical field based on their molecular weight only, we need to destroy the tertiary structure by reducing the protein to a linear molecule, and somehow mask the intrinsic net charge of the protein. That’s where SDS comes in.

The Role of SDS

SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or B-ME to break down protein-protein

disulphide

bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules. SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to its molecular weight. SDS is also present in the gel to make sure that once the proteins are linearized and their charges masked, they stay that way throughout the run. The dominant factor in determining an SDS-coated protein is it’s molecular radius. SDS-coated proteins have been shown to be linear molecules, 18 Angstroms wide and with length proportional to their molecular weight, so the molecular radius (and hence their mobility in the gel) is determined by the molecular weight of the protein. Since the SDS-coated proteins have the same charge to mass ratio, there will be no differential migration based on charge.

Sofia Origanti and Edwin Antony - 2015

The Gel Matrix

In an applied electrical field, the SDS-treated proteins will now move toward the positive anode at different rates depending on their molecular weight. These different mobilities will be exaggerated due to the high-friction environment of a gel matrix. As the name suggests, the gel matrix used for SDS-PAGE is polyacrylamide, which is a good choice because it is chemically inert and, crucially, can easily be made up at a variety concentrations to produce different pore sizes giving a variety of separating conditions that can be changed depending on your needs. The Discontinuous Buffer System and the Stacking Gel – Lining Them Up at the Starting LineTo conduct the current from the cathode (negative) to the anode (positive) through the gel, a buffer is obviously needed. Mostly we use the discontinuous Laemmli buffer system. “Discontinuous” simply means that the buffer in the gel and the tank are different.Typically, the system is set up with a stacking gel at pH 6.8, buffered by Tris-HCl, a running gel buffered to pH 8.8 by Tris-HCl and an electrode buffer at pH 8.3. The stacking gel has a low concentration of acrylamide and the running gel a higher concentration capable of retarding the movement of the proteins.

So what’s with all of those different pH’s?

Well, glycine can exist in three different charge states, positive, neutral or negative, depending on the

pH. This is shown in the diagram below. Control of the charge state of the glycine by the different buffers is the key to the whole stacking gel thing.

Sofia Origanti and Edwin Antony - 2015

Sofia Origanti and Edwin Antony - 2015

So here’s how the stacking gel works. When the power is turned on, the negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment, glycine switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the electric field. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine. The separation of Cl- from the Tris counter-ion (which is now moving towards the anode) creates a narrow zone with a steep voltage gradient that pulls the glycine along behind it, resulting in two narrowly separated fronts of migrating ions; the highly mobile Cl- front, followed by the slower, mostly neutral glycine front. All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl-, so when the two fronts sweep through the sample well, the proteins are concentrated into the narrow zone between the Cl- and glycine fronts.And They’re Off!This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them in the dust.The result is that the proteins are dumped in a very narrow band at the interface of the stacking and running gels and since the running gel has an increased acrylamide concentration, which slows the movement of the proteins according to their size, the separation begins.

What Was All of That About?If you are still wondering why the stacking gel is needed, think of what would happen if you didn’t use one. Gel wells are around 1cm deep and you generally need to substantially fill them to get enough protein onto the gel. So in the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep. Rather than being lined up together and hitting the running gel together, this would mean that the proteins in your sample would all enter the running gel at different times, resulting in very smeared bands. So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.

Once the proteins are in the running gel, they are separated because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins. The size of the pores in the gel can be altered depending on the size of the proteins you want to separate by changing the acrylamide concentration. Typical values are shown below. For a broader separation range, or for proteins that are hard to separate, a gradient gel, which has layers of increasing acrylamide concentration, can be used.

Sofia Origanti and Edwin Antony - 2015