Preparation of gene sequences for inserting them into a crop requires some parameters such as marker gene sequence. A marker gene sequence is one which can be detected at nearly every step of the transformation process. The detection process may be just physically and also for their actual expression. One of the most popular marke gene has been the gene for an antibiotic (e.g., ampicillin) resistance. Researches have been done to simplify and shorten the sequence of the process of genetic transformation for accuracy and predictability and efficiency. Transformation process has been simplified to such an extent that even such tests for structural or functional abilities of the gene to be transferred can be simply vies at the bacterial culture. A recent example is the Green Fluorescence Protein (GFP) gene extracted from genome of mutated jelly fish, Aequoria Victoria. The GFP mutant has the ability to survive and display enhanced bright green fluorescence, hence the name GFP. To the pure functionally competent sequence of this gene, another previously established marker gene for ampicillin gene has been attached. This compound gene has been effectively used for demonstration of the bacterial genetic transformation. This gene tagged with ampicillin resistance gene was obtained from a commercial supplier for bacterial transformation of E. coli HB101 K12. As publicized by the supplier and guided according t the suppliers instructions for its use except the step of using heat block at 42 0 C for 50 seconds (as a substitute for the use of water bath), there was no growth of any bacterial colonies.
This paper discusses the various plausible reasons for any repeated trials in the light of published information on the failures of the bacterial transformation process.
Genetic transformation is an accepted term for the use of molecular biology and genetic tools for altering an organism at the molecular level. Genetic transformation is a reality today. Virtually nearly any organism can be transformed genetically with a desirable disease curing, preventive and or resistant gene. The only prior requirement is that the DNA sequence of the desired should be extracted or constructed appropriately to suit the final objectives and should be easily transferable to the desirable recipient host species. This should be stable integrated in the host genome and should be made to express and function normally in the environment of the new genome of the recipient.
The present state of this knowledge is based in numerous researches carried out by Hayes, 1961 and several others and tons of published information is available on the methods, protocols, techniques, and their possible applications in molecular genetic improvement of animals, crops, beneficial microbes (e.g., Kosuge et al., 1983). Genetically transformed microbes have proven very valuable in large scale industrial production of insulin, many healthcare biomolecules, peptides, new generation antibiotics, and many more. They are potential in treating even the effluent areas as in the production of bioethanol as a renewable source of energy.
Basically deals with proven knowledge that purified DNA segments can enter into cells and get integrated into the nuclear material of the cells in such a manner that the new introduced fragment can even start functioning with normal expression. Thousands of DNA sequences have been characterized for their structure, location in the total nuclear DNA and their function. Now even complete genome sequences are established for man, major crops, Caenorhabditis elegans, yeasts and many bacteria. Such sequences have far reaching potentially useful implications in therapy of human diseases and infections, crop diseases and pests, and animals.
For all of these kinds of possible applications and implications many more directions of researches have produced results which gave been synthesized to realistically develop such workable schemes with nearly mathematical precision. A DNA sequence characterized is prepared and made ready for introduction into the host organism. A well known DNA sequence established for ability of gene promoting activity (called promoters) is attached. The so joined sequence of two different gene sequences is then attached to another gene sequence called the marker. These gene sequences are usually those which confer resistances to a range of antibiotics such a ampicillin, or nutrient (e. g., sugars such as arabinose) utilizing gene sequences.
In the present experiment, a gene for fluorescent green pigment characterized from a jelly fish was used to genetically transform Escherichia coli bacterium. The procedures used were standard but strictly as recommended by the commercial supplier of the gene. This gene was integrated into a plasmid called E. coli HB101 K12. This was obtained from a commercial supplier (BIORAD) who also provided the all the other required material along with detailed step by step instructions for carrying out the bacterial transformation with the gene for fluorescence.
The supplied plasmid, pGLO contained the GLO gene extracted and purified from a mutagen treated jelly fish called Aequoria victoria. The property of the GLO gene is to produce an enhanced bright Green Fluorescent Protein (GFP) which produced a bright green fluorescence when exposed to ultraviolet light in darkness. This plasmid also contained the ampicillin resistance (AMP) gene. The host bacterium containing this plasmid (pGLO) has the ability to grow on a nutrient medium to which a sugar arabinose has been supplemented. This medium also containing the antibiotic ampicillin whose presence does not prevent bacteria from growing normally. This antibiotic resistance gene helps and serves as a marker for easy direct method for single step selective preferential growth of the bacterium containing the GFP along with AMP. The suggested and the expected results were formation and growth white colonies on the nutrient medium with the added ampicillin and arabinose. The real transformed colonies have the ability to grow and display the characteristic bright green fluorescence on arabinose medium and under UV light illumination in dark. This experiment of bacterial transformation is an example of real life experience.
But in the present experiment, none of these results were observed. The possible problems and the probable reasons for the lack of growth of bacteria obtained from our transformation experiment are discussed.
Materials and Methods
All the materials used were supplied by BIORAD Labs. The glassware, equipments and other disposables were used as exactly recommended and suggested by the supplier in the accompanying Instruction Bulletin of the BIOTECHNOLOGY EXPLORER TM pGLO Bacterial Transformation Kit Catalog No. 166-0003EDU (explorer.biorad.com).
Materials and Methods
Kit Components Class Kit - 1. E. coli HB101 K 12, lyophilized in 1vial, 2. Plasmid (pGLO), lyophilized, 20 g in 1 vial, 3. Ampicillin, lyophilized, 30 mg in 1 vial, 4. L () Arabinose, lyophilized, 600 mg in1 vial, 5. Transformation solution (50 mM CaCl2, pH 6 sterile, 15 ml in 1 bottle, 6. LB nutrient broth, sterile, 10 ml 1 bottle, 7. LB nutrient agar powder, sterile (to make 500 ml) 1 pouch, 8. Pipets, sterile, individually wrapped 50 numbers, 9. Inoculation loops, sterile, 10 l, packs of 10 loops 8 pks, 10. Petri dishes, 60 mm in 2 sterile bags of 20 each, 11. 60 Microtubes, 2.0 ml, (10 each yellow, green, blue, orange, lavender, pink), 12. 8 Foam micro test tube holders, 13. Instruction manual 1
The transformation experiment required additional equipments.These were as follows and were present in our laboratory.
1. One UV lamplong wavelength (catalog 166-0500EDU) 2. One Clock to time 50 seconds 3. Microwave oven (1), 4. Incubator oven set to 37 0 C, 5. Temperature controlled water bath, 16 liter (1), 6. Thermometer that reads 42 0 C 1, 7. one 1 L flask , 8. 500 ml graduated cylinder (1), 9. Distilled water (from supermarket), 500 ml , 10. Crushed ice and containers (foam cups) (18)11. 10 ml of bleach (household variety) (10 ml), 12. Permanent marker pens (48)
a. Preparation of Nutrient Agar - All the LB nutrient agar from the kit was added to 500 ml of distilled water in a 1 L or larger Erlenmeyer flask. The flask was swirled thoroughly to allow full dispersal of the medium in water and placed in microwave oven for boiling stage with swirling the flask repeatedly 3 times in between. The flask was cooled to about 500 C on bench top for some time and then poured slowly into previously labeled Petri dishes. Agar plates were prepared 3 days prior to starting the experiment and were left for 2 days at room temperature before storing them in refrigerator until use.
b. Preparing arabinose and ampicillin solutions With a sterile pipetThree ml of sterile transformation solution (supplied) was added to arabinose containing vial and vortexed for thorough dissolution. Similarly 3 ml of the sterile transformation solution was added to vial containing ampicillin and mixed well. The vials placed such that the nutrient agar was not solidified.
LB nutrient agar prepared earlier was poured into Petri plates (about less than half) labeled as LB 16 plates, 16 as LBamp, and 8 plates LBampara. Then dissolved ampicillin was added to the medium and shaken this was poured into other 16 plates labeled LBamp. Similarly, to the rest of the LB medium arabinose was added and medium was poured onto 8 plates labeled as LB amp ara. These plates were stacked inverted in 4-8 numbers and stored in a refrigerator till use.
c. Preparing bacterial cultures and pGLO plasmid. E. coli bacteria (supplied as lyophilized) were rehydrated and allowed to stand at room temperature for 5 minutes. The petri plates were streaked in quadrants of Petri plates by wire loops with this inoculum to obtain single colonies. Similarly pGLO plasmid solution was prepared using the 250 ul transformation solution (supplied).
Transformation One ml of transformation solution (CaCl2) and 1 ml of LB nutrient solution were taken in the supplied color coded and labeled microtubes.
CaCl2 (250 ul ) was taken in two tubes each labeled as pGLO and pGLO-. A sterile wire loop was inserted into pGLO plasmid (fluorescing green in UV light). A loopful of this solution was taken and put into transformation solution (labeled pGLO). This was kept on ice for 10 min. No pGLO was added to pGLO- tube kept on ice.
With a new sterile loop bacteria from a single colony was collected and into inserted into the bottom of of the pGLO tube. Similarly another sterile loop was used to insert bacteria into pGLO- tube. The loops were spun thoroughly in the tubes so that the bacterial mass was fully dispersed without showing any clumps.
These tubes were placed back on ice.
With separate sterile pipets, 100 ul of each of these pGLO and pGLO- solutions were taken and uniformly skated on the surface of the appropriately labeled (LBamp plate pGLO LBampara plate pGLO other LBamp plate -pGLO the LB plate-pGLO) agar plates for even distribution of the solution on agar surfaces. The Petri plates were stacked and incubated in an oven at 37 0 C.
Heat Shock using the foam rack as a holder, pGLO and pGLO- tubes were placed into a water bath set to 42 0 C for precisely 50 seconds. After exactly 50 seconds, the tubes were shifted to ice very rapidly. The tubes were placed on ice for 2 minutes.
The foam racks containing the two tubes were brought to bench top at room temperature. Using a separate fresh sterile micropipettes 250 ul of LB nutrient broth was added to each tube. Each was flicked thoroughly to allow uniform distribution. These tube were incubated at room temperature for 10 minutes. Using fresh and separate micropipets 100 ul of each solution was dispensed into appropriately labeled plates (pGLO LB amp pGLO- LB amp ara -pGLO LB amp -pGLO LB. Using separately fresh and sterile loops the solution was evenly skated on the agar surfaces. The plates were stacked inverted and incubated in an oven at 37 0 C for overnight. The petri plates were observed the next morning.
The observations were on the growth of the colonies. Calculations were made on efficiency of transformation by using the counts of colonies formed, initial amount of DNA used. The colonies formed were also checked for fluorescence properties in the hand held UV lamp of long wavelength
All the necessary precautions were implemented in all the steps and stages in transformation. The transformed bacteria were incubated at 37 0 C for overnight. The only deviation from the recommended procedure was that instead of keeping the cultures in a water bath, they were placed in a heating block.
Surprisingly, there was no symptom of any growth of the transformed E. coli on the LB nutrient medium supplemented with the necessary concentration of arabinose as well as ampicillin. Therefore no data were available for carrying out the transformation efficiency and other paramerters.
It was disappointing to observe the unexpected results of no growth in the colonies of the E. coli transformed with the pGLO plasmid also containing beta lactamase gene for conferring ampicillin resistance. The experiment has to be repeated but with extra precautions to avoid any mistakes done earlier.
Many reasons and causes can be conceived. The procedure used in the present experiment deviated from the recommended one in the Instruction Bulletin in that the cultured bacteria were not placed in the water bath but placed in heat block. This may have caused too much of a sudden heat shock. It is well established that bacteria die beyond 420 C.
The other reason could be that the bacterial cells were not at the exact right stage (just before the log phase) for their readiness to be used to make them competent. Chemical methods of transformation are known to be not so efficient. Dury (2008) recommended the use of electroporation because chemical methods as used in the present experiment are not high enough to produce good desirable levels of transformation efficiencies. Similar suggestions were made by many other researchers on many other systems (e. g., Alegre et al 2004).
Importance of plasmid size and the DNA size may another important parameter for low efficiency of or no transformation. Szostkova and Horakova (1998) observed that the frequency of transformation is inversely related to the DNA and plasmid sizes.
Many other reasons may be assigned as follows
The plasmid may have been of poor quality
The plasmid may have been unstable under the conditions received and stored
A good and effecting of the heat shock may need to be checked again
Sterilizations methods used may have been very harsh
Incorrect or very antibiotics used
Preparations of the stocks and their final solution mixtures may not have been correct.
As no colony formation was observed the experiment needs to be repeated with extra precautions. Also the results as observed will also be communicated to the supplier for further advice and guidance on repeating the experiment.
In the repeat experiments more experiments have to be performed by using different transformation protocols including chemical and physical (electric) methods. In the chemical methods, it may be necessary to use different modifications of temperature levels, durations, the number of shocks. It may also be important and useful to use bacteria at different times during log and lag phases. This would help in catching the bacteria at the very time of their sensitivity and amenability to transformation methods.