BISC 219/F10: RNAi Lab 6

Lab 6: Series 3: Reverse Genetics- Cloning Your Gene of Interest

To see this process as an animation created by the DOLAN DNA center, go to | http://www.dnalc.org/view/15476-Genetic-engineering-inserting-new-DNA-into-a-plasmid-vector-3D-animation-with-with-basic-narration.html

Plasmids are circular pieces of DNA that can replicate in bacteria but are not part of the bacterial chromosome. Plasmids are generally circular molecules with fewer base pairs of DNA than the chromosome and with certain sequence elements (called the origin or ori) that allow the plasmid to replicate within the bacterial cytoplasm. Many naturally occurring plasmids have been modified for the purposes of using them as research tools. For example, a gene encoding resistance to an antibiotic can be added to a plasmid so that bacteria carrying the plasmid will become antibiotic resistant. This modification allows for selection of cells that carry plasmid DNA. A simplified map of the C. elegans RNAi plasmid is below:

To enable us to make lots and lots of RNA for RNA interference we need to express our gene at high levels. This is done with a specific strain of E. coli called HT115(DE3). The bacteria cells contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements. This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene. When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase. This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites. Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell. Additionally, this particular strain is deficient for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect! This E. coli strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transfomed cells to grow on media containing ampicillin.

Our goal is to insert our gene of interest into the pL4440 plasmid and transform HT115(DE) bacteria with the newly created plasmid.

Restriction enzyme digest of PCR product

To see an animation of this concept and process go to the Dolan DNA center at | http://www.dnalc.org/resources/animations/restriction.html

Once you have analyzed the agarose gel from last week and determined if the PCR amplification of your gene of interest was successful, you are ready to proceed to the next step: Restriction Enzyme Digestion of the amplified DNA.

What do restriction enzymes/endonucleases do? They are enzymes have been isolated from bacteria and cut single or double stranded DNA at specific recognition sequences. These recognition sequences are often palindromic (read the same forwards and backwards). The purpose of these enzymes in bacteria is to eliminate foreign DNA that enters the cells (ie bacteriaphage genomes) to protect the host genome. Companies now purify these enzymes from the bacteria and we can use them to manipulate DNA to link different DNA strands together.

There are two kinds of "cuts" a restriction enzyme can make, either blunt ended or overhangs called "sticky" ends. The blunt ended cuts cause the DNA to have no single stranded "overhangs" that can facilitate base pairing with another strand of complimentary DNA. This makes joining two pieces together harder but it can be done. The "sticky" ends make joining two pieces together with complimentary base pair overhangs much easier to do and is much more likely to happen during a ligation reaction.

For more information on restriction enzymes you can read the information on Wikipedia or my favorite site for restriction endonuclease information New England Biolabs. At NEB you can click on the different enzymes and look at the information they have available about the recognition sequence for cutting, the conditions for effectiveness and lots more.

Protocol for RE digest

1. Obtain your PCR sample from last week from your instructor.
2. Pipette 10 ul of your PCR into a clean 1.5 ml tube.
3. Add 2 ul of 10X NEB restriction buffer 2 to the tube.
4. Add 1 ul of BSA (bovine serum albumin) if working with bli-1 or vab-10.
5. Add 1 ul of enzyme 1
6. Add 1 ul of enzyme 2
7. Bring the volume of your reaction to 20 ul total with dH₂O
8. Incubate the reaction at 37°C for 45 minutes to allow for proper digestion.

For rol-5 the enzymes you are cutting your DNA with are BglII and HindIII. For bli-1 you are cutting with BglII and SpeI.

Your instructor will have already cut and purified the pL4440 vector for you. You will need the vector for the ligation reaction.

Purification of samples (remove enzymes)

In order for your ligation and transformation of your new contruct to happen you MUST remove the enzymes from the reaction. There are a few ways of doing this. One is to denature the enzymes with heat, although not all enzymes can be "heat killed". Thus the better option is to purify the DNA away from the enzymes (proteins) and there are wonderful kits out there to do this in a few easy steps. We will use the QIAquick PCR Purification Kit from Qiagen.

We will follow the manufacturer's instructions:

1. Obtain a collection tube and insert (contains the silica matrix) from your instructor.
2. Make sure the insert is in the collection tube.
3. Add 250 ul of buffer PB to the insert.
4. Add your entire 20 ul restriction enzyme digest sample to the insert + buffer.
5. Invert a few times to mix.
6. Centrifuge your sample for 1 minute at 13,200g.
7. Discard the flow through in the collection tube, put your insert back into the collection tube once its empty.
8. Add 700 ul of Wash Buffer (contains EtOH) to your insert.
9. Centrifuge for 1 minute at 13,200g
10. Discard the flow through in the collection tube, put your insert back into the collection tube once its empty.
11. Centrifuge for 1 minute at 13,200g again. This removes any residual wash buffer that would contaminate your final elution.
12. Discard your collection tube in the waste bucket.
13. Put you insert into a clean 1.5 ml tube.
14. Add 25 ul of elution buffer to the center of the silica matrix without touching it.
15. Centrifuge for 1.5 minutes
16. Discard the insert and label your final sample with your initials and what is in the tube.

Ligation into the pL4440 vector

To see an animation of this process go to the Dolan DNA center at | http://www.dnalc.org/view/15541-DNA-ligase-joining-two-lengths-of-DNA-at-their-sticky-ends.html

You have now "cut" the ends of your PCR product to make them amenable to being joined or ligated back together with other DNA molecules cut with the same restriction enzyme to make complementary base pairing possible. This is a very common practice in molecular biology to "clone" or insert genes or pieces of genes into plasmids. We will be using an enzyme called T4 DNA ligase from bacteriophage T4. For more information about ligases see Wikipedia or our exact enzyme from NEB.

Ligation Protocol:
For an effective ligation you want an excess of stick end inserts - typically the smaller piece of DNA to the plasmid, the larger piece of DNA.

We will do a 20 ul reaction.

1. Add 2 ul of 10X T4 DNA Ligase Reaction Buffer to a 0.5 ml tube - what is the final reaction concentration?
2. Add 3 ul of pL4440 that your instructor digested.
3. Add 9 ul of insert.
4. Add 1 ul of T4 DNA ligase.
5. Add 5 ul of dH₂O
6. Incubate the reaction at room temperature for 30 minutes.

Proceed to the transformation.

Transformation into competent cloning cells

Here are two animations from the Dolan DNA center that describe the history and the process of interspecies incorporation and expression of DNA (transformation): | http://www.dnalc.org/resources/animations/transformation1.html AND | http://www.dnalc.org/resources/animations/transformation2.html

During “transformation,” a single plasmid enters a single bacterium and, once inside, replicates and expresses the genes it encodes. In this case, the relevant genes expressed are for ampicillin resistance and for the piece of the C. elegans gene of interest. The transformation mixes were given a short time to express these gene products and then were spread on an agar plate that contained nutrients and the antibiotics tetracyclin (encoded by the bacteria) and ampicillin (encoded by the plasmid). Only the cells that incorporated the plasmid DNA and expressed the plasmid genes grew to form colonies of bacteria in the presence of ampicillin. The untransformed bacteria failed to form visible colonies on the ampicillin containing agar surface.

Most bacteria do not usually exist in a “transformation ready” state, but the bacteria can be made permeable to the plasmid DNA by exposing them to calcium chloride. Cells that have been treated with calcium chloride or are otherwise capable of transformation are referred to as “competent.” Competent cells are extremely fragile and must be handled gently, i.e. kept cold, not vortexed, etc. The transformation procedure is efficient enough for most lab purposes; with efficiencies as high as 10⁷ transformed cells per microgram of DNA, but it is important to realize that only 1 cell in about 10,000 is successfully transformed.

This is especially true for "cloning competent" bacteria. The newly ligated plasmids are few in number compared to the number that can be isolated during a miniprep (you will do this more efficient isolation in a future step) so the cells are made "ultra-competent" and usually purchased from a company for A LOT of money - around $300 for 20 transformations! The newly ligated plasmid DNA is not as tightly wound as one isolated from a cell so the plasmids themselves are fragile and can be sheared and rendered untransformable. Please be gentle with your cells and your newly formed plasmids!

Transformation of newly ligated plasmid DNA into BL21 cells
The BL21 bacterial cells are on the instructor’s bench You will transform some of your plasmid DNA into this strain. The cells are very fragile, so treat them gently.

1. Label the top or the side of the tube with BL21, pL4440 (your gene name), and your initials or team color.
   
2. Start the transformations by pipetting 25 microliters of well mixed (gently!) chemically competent cells to the labeled microfuge tube prepared in the the last step.
   
3. Add 5 microliters of your ligated plasmid DNA to the tube. Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit on ice for 10 minutes.
   
4. Heat shock by incubating the transformation mix at 42°C for 60 seconds, exactly. This step must be timed exactly. Remove the tubes at the end of 60 seconds to your ice bucket while you get your LB ready.
   
5. Pour 3 mls LB from the stock bottle into a clean and sterile 15 ml conical tube. By doing this, you will minimize the amount of LB that will be contaminated if you accidentally touch the media with something that is not sterile. Contaminated media looks cloudy, so be sure to swirl and examine the stock bottle of LB to make sure it is not contaminated.
   
6. Add 500 microliters of the LB in your conical tube to the transformation mix. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip.
   
7. Once you have added the media, close the cap and invert the tube once or twice to mix the contents. Incubate at 37°C for 45 minutes.
   
8. While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label one LB + amp agar plate. Label the bottom of the plate with the strain's identity (BL21), the plasmid used (your unique plasmid name), the date, your initials and team color. You must label the bottom of the plate since the tops are easily switched. Put this plate in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.
   
9. Once the transformation mix has incubated at 37°C for 45 minutes, invert it to mix the contents and pipet 100 microliters of transformed cells onto the center of that slightly dehydrated LB plate prepared in the previous step. Pour 5-10 glass beads onto the plate. Put the lid back on and gently swirl the bead all over the plate to spread the transformed bacteria around. When you are done - pour the beads into the beaker on the bench.
10. Replace the lid and leave the agar plate undisturbed for a few minutes.
11. Once it has dried enough that the surface doesn’t appear wet, invert the plate and incubate them at 37°C for 24 hours. The plate should be incubated with the agar side up so that condensation will not drip onto the surface of the agar and smear the colonies that will be growing there.
12. Save the remaining transformation mix for 24 hours or until we are sure that there is at least one colony growing on each of your plates.
    

What would it mean if you had no colonies on your plate? Normally, you would expect to have around 10-100 pale color colonies on each plate. After the 24 hour growth period the plate should be placed in the rack in the refrigerator labeled with your lab day. If you have no colonies on one or more of your plates, please notify your instructor right away.

Before leaving lab today, give the rest of your ligated plasmid DNA to your instructor in a labeled microfuge tube. Make sure your tube is labeled with your name, lab day, plasmid name and color coded with a piece of tape in your team color.

Outline of Experimental Design for REVERSE Genetics Project

Where are you now in this process?(What have you done so far; What's next?)
Make the feeder strain of bacteria

1. Amplify gene of interest by pcr ;
   
2. Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
   
3. Clean up DNA (remove enzymes);
   
4. Cloning: ligate gene into vector plasmid with amp resistance gene ;
   
5. Transform competent bacterial cells of a strain genetically modified to be tetracycline resistant;
6. Select for transformants on media with tetracycline and ampicillin;
   
7. Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
8. Culture the selected colony from colony pcr to create a lot of copies of these bacteria
9. Isolate the cloned plasmid DNA from that cultured colony by miniprep;
   
10. Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
    
11. Select for transformants on media with tetracycline and ampicillin
12. Choose an isolated colony to culture and make lots of feeder strain bacteria;
    
13. Seed NM lite worm growth media plates with feeder strain produced as described
    

Plate wild type C. elegans worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).

Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that we NOT fed feeder strain bacteria.

Isolate RNA from RNAi worms and control worms of same strains.

Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.

Visualize cDNA in the pcr product by agarose gel electrophoresis and compare size of amplified fragment to known size of coding regions of gene of interest.

Assignment

Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.

Links to Labs& Project Info

Series1:
Worm Info
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Series2:
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Finish Complementation; Mapping Con't
Lab 6: DNA sequence analysis; Mapping Con't
Lab 7: Complete Mapping: Score
Series3:
Schedule of Reverse Genetics Project
RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification
Lab 11: RT PCR reactions