Bacterial Transposons GeNei' Objectives: * To understand the process of Bacterial Transposition by mating 2 strains of bacteria and + To analyse the effect of transposition in a given Bacterial strain Principle: Transposons are ‘Transposable elements’ which are able to move from one place to another within a cell's genome. Sometimes a copy is made and the entire copy moves. This kind of insertion requires target DNA sequences. In the process they may cause mutations, increase or decrease in the amount of DNA in the genome, promote gene rearrangements, regulate gene expression or induce chromosome breaks or rearrangements. These mobile segments of DNA are sometimes referred to as ‘Jumping genes’. The firet Transposable elements (TEs) were discovered in maize (Zea mays), by Barbara McClintock in 1948, for which she was awarded a Nobel Prize in 1983. She noticed insertions, deletions, and translocations, caused by these elements which lead to a change in the color of corn kernel. There are two distinct types of transposons: DNA Transposons, consisting only of DNA that moves directly from one place to place and Retrotransposons, which first transcribe DNA into RNA and then use Reverse Transcriptase to make a DNA copy of the RNA to insert ina new location In bacteria, transposons can jump from chromosomal DNA to plasmid DNA and back. They usually an additional gene for function other than transposition-usually an antibiotic resistance gene cary Bacterial transposons of this type belong to the Tn family. When the transposable elements lack and additional gene they are called Insertion sequences. (© Merck Specialties Private Limited, 2013Bacterial Transposons. GeNei™ (a) ae (©) Fiokking OA ml oeneroes) “Tr Iermediate mobic clomer Fig 1: Classification of transposons Fig 2: Mechanism of transposition (© Merck Specialities Private Limited, 2013Bacterial Transposons GeNei' Bacterial transposons are also classified into 4 types: 1. Insertion sequence (IS): Most of the sequence is taken by one or two genes for Transposase enzyme which catalyzes the transposition. IS elements transpose either replicatively or conservatively. Eg.IS1 and S186, present in the 50kb segment in the E.coli DNA. A single E.coli genome may contain 20 of them. 2. Composite transposon: Bacteria contain composite mobile genetic elements that are larger than IS elements and contain one or more protein coding genes (often for antibiotic resistance) in addtition to those required for transposition. They use conservative method of transposition. 3. Tn3 type transposon: These are usually 5000bp long, code for Transposase, B-lactamase and Resolves which controls Transposase thereby decreasing recombination in transposon. 4, Transposable Phages or bacterial viruses, which transpose replicatively as part of their normal infectious cycle. These integrate into the E.coli chromosome at the regulatory element. Based on the mechanism of transposition, bacterial transposons are classified as conservative or replicative. In replicative transposition, there is direct interaction between donor and target site, resulting in copying of donor element. Conservative or non-relicative transposition involves the excision of the element and re-integration in a new site Transposons are found in all major branches of life and have spread by horizontal gene transfer and have greatly contributed to evolution through duplication and chromosomal re-arrangements. One family of TEs in the fruit fly Drosophila ‘melanogaster are called P elements. The most common form of transposable element in humans is the Alu sequence. Transposons are a useful tool in plant molecular biology. Researchers use it as a means of mutagenesis to identify mutant alleles, to study chemical mutagenesis methods, to study gene expression etc. 3 (© Merck Speciaitos Private Limited, 2013Bact Transposons GeNe: Kit Description: Using this kit, students can learn the process of transposition occurring naturally between two strains. Two E.coli strains are provided of which Strain A is resistant to Ampicillin and Chloramphenicol. Then Ampicillin Resistance Marker is present on a transposable plasmid. Strain B is resistant to streptomycin. When these two strains are grown together in appropriate conditions, Ampicillin resistance gene from one strain will get transferred to the other making it resistant to ampicillin along with streptomycin. This can be observed by the growth of the recipient on ampicillin plate and also by colony morphology. To confirm the transfer of the ampicillin resistance gene, DNA miniprep (Plasmid Isolation) is carried out 616112400011730 : The kit is designed to cary out 5 transposition experiments using single lyophilized vial of two E.coli straine. 616112400021730 : This kit is designed to carry out 20 transposition experiments. Two lyophilized vials of each E.coli strains are supplied, each for 10 transposition experiments Duration of Experiment: Experiment is carried out over a span of 6 days, approximate time taken on each day is indicated below: Day1 2 hours (Preparation of media and revival of host) Day 2 : 30 mins (Inoculation) Day 3 : 11 hours (Growing, mixing and incubation) Day 4 : 4 hours (for media preparation and for plating) Day 5 : ‘thour (Observation) Day 6 : 5 hours (Miniprep, electrophoresis, observation and interpretation) 4 © Merck Specialties Privat Limited, 2013Bact ial Transposons GeNe: Materials Provided: The list below provides information about the materials supplied in the kit. The components should be stored as suggested Gant Materials GTSTIRAOOOTITIO | GISTIZAGOOZTTIO | Store (SExpts.) (20 Expts) E.cof Strain A 1 Vial 2 Vials [4° E.coli Strain B 1 Vial 2 Vials [4° Chloramphenicol 20mg 2x30mg | 4°C ‘Ampicilin 700 mg 3x100mg_| 4°C ‘Streptomycin 100 mg 2x150mg_| 4° Solution | 2 mi 10m! | 4°C Solution 1 4 mi 15mi__| 4° Solution ti 3 mi 10mi__| 4°C ‘Solution 1V 70 mi 40mi__| 4°C 2.5X Gel o Loading Butfer 0.25 mi 0.25 mi ac RNase A OA mi o3mi__| eC 1X TE 0.5 mi 2m aC ‘Agarose 259 70g RT '50X TAE 20 mi 80mi_|_RT LB Broth 50g 200g RT ‘Agar 50g 100g RT 4.5 mi Vials 50 Nos 3x50 Nos [RT Note: Room Temperature (RT) not to exceed 20-25°C Materials Required: Equipment : Centrifuge, Shaker Incubator (37°C), Static Incubator Glassware : Test tubes, Conical Flasks, Petri Plates, Measuring Cylinder Reagent : Distilled Water, 70% Ethanol Other Requirements: Micropipettes, Crushed Ice, Tips 5 (© Merck Specialties Privat Limited, 2013,Bacterial Transposons GeNe Note : * Read the entire procedure carefully prior to starting the experiment. * All microbiological operations should be done strictly under aseptic conditions. * Revive the strain as soon as the lyophilized vial is opened. + Carry out the experiments within 2 weeks of reviving the strain. * Use sterile water for Ampicillin, Streptomycin and 70% ethanol for Chloramphenicol preparation. «Thaw solution II before use. * For preparation of gel refer Agarose Gel Electrophoresis. (Page no: 12) + Ensure that no moisture is present on the surface of the hard agar plates. * For preparation of media, antibiotics refer appendix. (Page no: 11) © LB atoocas: LB with 100ughnl of Ampicillin and 20u1g/mi of Chloramphenicol. + LB si1oo: LB with 100yg/ml of Streptomycin. * LB astoosiioc: LB with 100ug/mi of Ampicillin and 100yg/mI of Streptomycin. * LB sttoocao: LB with 20ug/ml of Chloramphenicol and 100ug/m! of Streptomycin. Procedure: Day 1: Revival of Host 1 2. Break open the lyophilized vials of two E.coli strains A and B and resuspend them in 0.1 ml of LB broth. Streak a loopful of Strain A on LB plate with 100 pg/ml of Ampicillin, 20 ug/ml of Chloramphenicol. Strain B on LB with 100 g/ml of Streptomycin and inoculate the remaining suspension in 5 ml LB broth with respective antibiotics. Incubate the plates and tubes at 37°C for 16 hours. 6 (© Merck Specialties Private Limited, 2013,Bacterial Transposons GeNe: Day 2: 1. Inoculate a single colony of both Strain A and Strain B from the respective plate into 5ml LB tube with Ampicillin, Chloramphenicol (Strain A) and Streptomycin (Strain B).. 2. Incubate in a shaker incubator at 37°C for 16 hours at 160 rpm. Day 3: 1. Inoculate 0.2 ml of overnight broth of both Strain A and ‘Strain B into 5 mI LB broth with respective antibiotics. Incubate strain A for 4 hours and strain B for 3 hours at 37°C. 3. Take 0.2 ml of both Strain A and Strain B in a tube. 4. Incubate at 37°C for 3 hours (Static incubation). 5. Then add 2ml of LB Broth. 6. 7. N . Incubate at 37°C for 4 more hours (static). Leave the tubes at 4°C for further use. Day 4: 41. Remove the tube (Day 3 step 7) from 4°C and allow it to come to room temperature. 2. Plate 100pl of Strain A, Strain B (Day 2 step 2) and the mixture of both (which was kept at 4°C Day 3 step 7) on the following combinations of LB plates.LB arcostioo, LBst100, LBarooca0 and LB ss1o0c20. 3. Incubate the plates at 37°C for 16 hours. Day 5: 1. Observe the plates. 2. If plates with Strain A + Strain B mix do not show colonies, then incubate the plates further at 37°C for 16 hours. 3. Leave Strain A and Strain B plates at 4°C Day 6: 1. Observe the growth of Strain A and Strain B mix on LB plates with all 4 combinations of _ antibiotics. (Day 4 Step 2). 7 (© Merck Specialties Private Limited, 2013Bacterial Transposons GeNei™ * Strain A will grow on LB atooc2o and Strain B will grow on LB siico but both will not grow on LB aroosi100 oF LB st100c20 * A mix of both Strain A and Strain B will grow on LBatoostio, indicating the transfer of gene from one strain to the other. This mix of Strain A and Strain B will not grow on LB sttooc20 thus showing the transfer of only Ampicillin gene from the transposable plasmid but not the Chloramphenicol gene, which is present on the other plasmid. * This transfer of gene from one strain to the other can be confirmed by doing a DNA miniprep or plasmid isolation from all the strains (Strain A, Strain B and the mix of both) Protocol for isolotion of plasmid DNA (miniprep): 1. Take a loop full of Strain A and Strain B from the plates (revived plates of Day 2 step 2 or Day 5 step 3 plates) and resuspend in 0.5 ml of LB Broth 2. Similarly take a loop full of Strain A and Strain B mix from LB ascosi100 plate (Day 6 step 1) and resuspend in 0.5ml LB Broth. 3. Centrifuge the vials, remove the supernatant without disturbing the pellet. 4, Resuspend the pellet in 100ul of Solution I, vortex, keep on ice for 5 minutes and then at room temperature (RT). 5. Add 200 pl of Solution Il at room temperature (RT). Mix gently by inverting the vial five times (do not vortex). 6. Add 150pl of Solution Ill. Mix gently by inverting the vial. Place on ice for 5 minutes. 7. Centrifuge at 6000-8000 rpm for 10 minutes at 4°C. 8. Transfer the supernatant immediately to a fresh vial and add 450 ul of Solution IV to precipitate the DNA. Mix by inverting the vial. Incubate at RT for 10-15 minutes. (© Merck Specialties Private Limited, 2013,| Transposons. le: 9. Centrifuge at 10,000 rpm for 20 minutes or at 6000 rpm for 30 minutes. Decant the supernatant, invert the vial on blotting paper to drain out left over supernatant, 10. Dry the DNA pellet at 37°C for 10-15 minutes til there is no trace of Solution IV. 11: Resuspend the pellet in 20 of 1X TE (add along the sides). Mix by tapping the vial so that DNA goes into the solution. 12. Add Sul of RNase to the vials and leave at room temperature for 10 minutes. 13, Meanwhile prepare 1% agarose gel (Refer Agarose gel electrophoresis page 12) 14, Add 2ul of Gel Loading Buffer to each of the samples and load 20 ul in the wells of the 1% agarose gel Electrophorese at 100 volts for 1-2 hours. 15. Visualize the bands under a UV Transiluminator. (© Merck Specaiies Private Lined, 2013Bacterial Transposons GeNei’ Observation: Observe the bands in all three strains. Interpretation: * On analyzing the bands after electrophoresis, one observes two plasmid DNA bands in Strain A and no comparable bands in Strain B and the mix. ‘+ The two bands in Strain A correspond to two plasmids one is transposable plasmid with Ampicillin marker and the other plasmid with Chloramphenicol marker. * Strain B does not show any band indicating the absence of any plasmid. Similarly mix of both Strain A and Strain B does not show any band Note: Sometimes genomic DNA contamination can be seen as a faint band at the top. * Thus it shows that only Ampicillin gene has got transposed from E.coli Strain A to Strain B making it resistant to Ampicillin, and this gene has got inserted in the chromosome of Strain B. Lane 1: Strain A Lane 2: Strain B Lane 3: Strain A + Strain B Mixture Fig 3: Analysis of DNA from the two E.coli strains showing Transposition of the Ampicillin gene from Strain A to Strain B 10 (© Merck Specialities Private Limited, 2013Bacterial Transposons GeNei™ Appendix: Preparation of LB agar/broth (1 litre): Dissolve 25g of LB broth media in 800m! of distilled water. Adjust the pH to 7.0 with 5N NaOH (if necessary) and make up the volume to 1 litre. Sterilize by autoclaving. For LB agar, add 1.5%agar to LB broth and autoclave. Preparation of Antibiotic Solutions: Chloramphenicol: Reconstitute Chloramphenicol in 70% ethanol to get a stock concentration of 20 mg/ml as follows * 201mg in 1 ml of 70% ethanol + 30 mg in 1.5 ml of 70% ethanol. Store at 4°C. Use the reconstituted antibiotic solution within 2 weeks. Ampicillin: Reconstitute 100 mg of Ampicillin in 1ml of sterile water to get a stock concentration of 100 mg/ml. Store at 4°C. Use the reconstituted antibiotic solution within 2 weeks. Reconstitute Streptomycin in sterile water to get a stock concentration of 100 mg/ml as follows: * 100 mg in 1 ml of sterile water * 150 mg in 1.5 ml of sterile water Vortex if necessary. Cover the vial with aluminium foil as it is light sensitive and store at 4°C, Use the reconstituted antibiotic solution within 2 weeks. For Antibiotic containing LB media: Add Chloramphenicol to LB broth or agar al a final concentration of 20 yg/ml, when the temperature of the medium is around 40-45°C. Similarly add Ampicillin and Streptomycin to LB broth or agar at a final concentration of 100ug/ml, each when the temperature of the medium is around 40-45°C. Note: Do not add antibiotics to very hot medium. "1 © Merck Speciaties Private Limited, 2019,Bacterial Transposons GeNei™ Agarose Gel Electrophoresis 12 (© Merck Spociaiios Prva Limited, 2013,Bacterial Transposons GeNei™ Introduction: Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology. The technique consists of 3 basic steps: © Preparation of agarose gel * Electrophoresis of the DNA fragments ‘* Visualization of DNA fragments Principle: Preparation of Agarose Gel: Agarose is a linear polymer extracted from seaweeds. Its basic structure is shown in figure. H cH. 9 QO. Bs Figure: Agarose structure unit Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution of agarose is then poured into a mould and allowed to solidity. As it cools, agarose undergoes polymerisation i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose 13 © Merck Specialities Private Limited, 2013Bacterial Transposons GeNei™ Electrophoresis of DNA fragments: Electrophoresis is a technique used to separate charged molecules. DNA is negatively charged at neutral pH and when electric field is applied across the gel, the DNA migrates towards the anode. Migration of the DNA through the gel is dependent upon: 1. Molecular size of DNA 2. Agarose concentration 3. Conformation of DNA and 4. Applied current Matrix of agarose gel acts as a molecular sieve through which DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces. While lower concentration of agarose helps in the movement of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are Xylene cyanol and Bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These tracking dyes are negatively charged, low molecular weight compounds that are loaded along with each sample at the start of electrophoresis, when the tracking dye reaches towards the anode, electrophoresis is terminated. 14 (© Merck Specialities Privat Limited, 2013Bacterial Transposons GeNei™ Visualisation of DNA fragments: Since DNA is not naturally coloured, it will not be visible on the gel. Hence, the gel after electrophoresis, is stained with a dye specific to the DNA. Discrete bands are observed when there is enough DNA material present to bind the dye to make it visible, otherwise the band is not detected. The gel is ‘observed against a light background wherein DNA appears as dark coloured bands. Alternatively, an intercalating dye like Ethidium bromide is added to agarose gel and location of bands determined by examining the gel under UV light, wherein DNA fluoresces. Note: + Ethidium bromide must be handled carefully as it is a mutagen and a carcinogen. + Gels with Ethidium bromide are to be collected in separate plastic bags and shipped to an external waste management agency for incinetation as part of hazardous solid waste. 15 (© Merck Specialtios Private Limited, 2013Bacterial Transposons GeNei™ Procedure: Preparation of 1% Agarose Gel 1 Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer. (For one experiment, approximately 200 mi of 1X TAE is required. Dilute 4 ml of SOX TAE to 200 mi with distilled water). Weigh 0.5 g of agarose and add to 50 ml of 1X TAE. This gives 1% agarose gel. Boil till agarose dissolves completely and a clear solution results. Add 2 jl Ethidium bromide to the molten agarose (from a stock of 10 mg/ml in water), when the temperature is around 55°C. Mix and cast the gel Meanwhile place the combs of electrophoresis set such that it is approximately 2 cm away from the cathode Pour the agarose solution in the central part of tank when the temperature reaches approximately 55°C. Do not generate air bubbles. The thickness of the gel should be around 0.5 to 0.9 cm. Keep the gel undisturbed at room temperature for the agarose to solidify. 16 (© Merck Speciaities Private Limited, 2013Bacterial Transposons GeNei™ Electrophoresis 7. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 om above the gel surface. 8. Gently lift the comb, ensuring that wells remain intact. 9. Connect the power cord to the electrophoretic power supply according to the convention red: anode, black: cathode. 410. Load the samples in the wells in the desired order. 411. Set the voltage to 50 V and switch on the power supply. 12. Switch off the power when the tracking dye (bromophenol blue) from the well reaches 4.5 cm of the gel. This takes approximately one hour. 43.After electrophoresis, DNA samples can be visualized under UV light. They appear fluorescent. 7 (© Merck Specialities vate Limited, 2013,Bacterial Transposons GeNei™ Ordering Information: Product Size Cat# GeNei™ Bacterial Transposons 1EA 616112400011730 Teaching Kit Consumables for 5 Experiments) GeNei™ Bacterial Transposons 1EA 616112400021730 Teaching Kit Consumables for 20 Experiments) Sales: [email protected] Customer Suppo! [email protected] 18 (© Merck Spocaiies Private Limted, 2013