Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi
Through reverse genetics we will deduce the function of a gene starting with its sequence and working back to its phenotype. There are many genes in the genome whose phenotype when mutated is lethal; therefore, it's impossible (or very difficult) to tie function to a particular gene in the traditional forward genetics manner of creating random mutations, looking for phenotype changes, and then finding the defective gene responsible for that function.
In our reverse genetics study of some interesting C. elegans genes, two different strains of worms, wild-type and rrf-3 (RNAi enhanced), are fed bacteria expressing dsRNA specific to a particular worm gene. Ingesting dsRNA initiates cascade of events that leads to the destruction of the mRNA of the target gene. An altered phenotype in the progeny of RNAi-treated worms indicates what happens when the normal function of this gene is lost or significantly downregulated.
Double stranded RNA (dsRNA) can be introduced to the C. elegans cells in many different ways including: feeding, injection and soaking. Each of these methods has positives and negatives. We are using the feeding method - where we use genetically modififed bacteria as dsRNA factories.
To begin to investigate the power of reverese genetics, you will need to grow your own induced bacteria to seed your plates for RNAi feeding.
Done on the night before this lab:
You and your partner made an overnight broth culture of your selected colony. This process created a sub-culture of many identical copies of the plasmid carrying the construct that will induce RNAi to silence or downregulate the gene that you want to study.
On the morning of lab:
Your instructor or the lab staff will come in early in the morning and sub-culture your bacterial overnight. The cells will be in stationary phase in the morning and sucessful induction requires log phase growth.
To create the subculture of bacteria your cultures will be diluted 1:10 (500 μL of culture into 4.5 ml of LB + amp). These cultures will be allowed to grow until lab time - approximately 3-4 hours.
When you come in to lab you will induce your cultures to make lots of dsRNA by adding IPTG to the culture and letting it continue to incubate for a few hours so the cell is full of dsRNA. The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein. The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our C. elegans DNA into RNA!
A simplified map of the C. elegans RNAi plasmid :
More specifically: The bacteria cells of strain HTll5(DE) 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. The IPTG induction allows us to "turn on" and express the plasmid gene of interest only when we want to and it allow us to make much higher levels of RNA for RNA interference than would be made without this induction.
Another useful thing about E coli strain HT 115(DE)is that 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 upregulate production of dsRNA of our worm gene of interest in pL4440 vector plasmids containing that gene in HT115(DE) bacteria.
To induce your cultures:
- Add 5 μL of 0.5 M IPTG to your culture. What is the effective concentration of IPTG in your culture?
- Put your culture back in the 37°C incubator in the spinning wheel for approximately 3 hours.
- After the lab introduction, we will head up to the penthouse to discuss the papers you were assigned.
To do after induction is complete:
- Pour your culture into a 15 ml orange cap centrifuge tube.
- Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
- Remove 3.5 ml of the supernatant.
- Resuspend the bacterial pellet in the remaining 1.0 ml of supernatant - you are concentrating your bacteria.
- Pipet a 200 μL aliquot of your induced bacteria onto the center of 4 feeding plates. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.
- Allow the bacteria to be absorbed into the media
- Obtain 2 control plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, except that the pL4440 plasmid is only expressing RNA from the vector - it lacks DNA specific to any worm genes.
- Stack all 6 plates and wrap with an elastic - put them in the lab box with a piece of your tape on top.
- We will allow the bacteria to continue to induce overnight at room temperature.
4 days before next lab:
- Come into lab and find your stack of plates.
- On 2 of the experimental plates add 2 L4 wild type (N2) hermaphrodites
- On 2 of the experimental plates add 2 L4 rrf-3 hermaphrodites
- On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites
- On 1 of the control plates add 2 L4 rrf-3 hermaphrodites
- Wrap all of your plates in an elastic and stick in your lab day box in the worm incubator set at 23°C
You will score your phenotypes in the next lab.
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
- Amplify gene of interest by pcr ;
- Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation;
- Clean up DNA (remove enzymes);
- Cloning: ligate gene into vector plasmid with amp resistance gene ;
- Transform competent bacterial cells of a strain genetically modified to be tetracycline resistant;
- Select for transformants on media with ampicillin;
- Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
- Culture the selected colony from colony pcr to create a lot of copies of these bacteria
- Isolate the cloned plasmid DNA from that cultured colony by miniprep;
- Retransform isolated plasmids (with gene interest) into ITPG inducable HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;
- Select for transformants on media with ampicillin
- Choose an isolated colony to culture and make lots of bacteria;
- Induce expression of C. elegans gene dsRNA from pL4440 vector by IPTG induction of log phase culture of HT115(DE) E. coli
- Seed NM lite worm growth media plates with induced feeder strain bacteria
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.
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
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
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
Schedule of Reverse Genetics Project
RNAi General Information
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