12.12.2014
While looking for a project for this year’s science expo (or science fair as it is called in the States), I came across the most fascinating thing. Being the skeptic that I am I desperately wanted to find out if UV light can actually be used as a sterilization tool. A bit more on yeast then:  Yeast is part of the fungus family and when yeast has something to feed on like sugar and also has oxygen, it buds. Our method was as follows: In order to culture the yeast we mixed it in some sugar water, put it into 3 glass bottles and left it in  a dark cupboard for 2 days.
We observed that even on the 20 minute exposure the yeast cultures were thriving however we did notice that the growth was   substantially less than what we found on the plates that had been exposed for shorter periods.
You can move the ROI away from the center of the field of view to avoid the glare from the light. It is unclear how sensitive this process is to the dormant cultures from the refrigerated plate.
In this lab, purified DNA from bacteriophage lambda was digested with a series of restriction enzymes, resulting in the cleavage of the DNA strand into portions of various lengths. Results of the restriction analysis were close to those given as the "ideal gel" in the text. Lab protocol called for the creation of two cultures, one incubated with plasmid containing GFP and an ampicillin resistance gene (+), the other without (-). However, no colonies were visible on the ampicillin plates, indicating that transformation was unsuccessful.
Visualization of the plasmid-transformed plates under UV light confirmed the preliminary assessment. The failure of the transformation was most likely due to an error in executing the laboratory procedure. The experiment is repeated with 10 uL of plasmid at different concentrations as shown below. Mass of DNA spread appears to increase the number of colonies observed until the response saturates. Beta-lactamase kills a carbon from the penicillin molecules, producing so-called penicilloic acid and hydrogen ions.
Increasing the concentration of either beta-lactamase or penicillin-G would speed the reaction; the reaction is caused by interaction of these molecules, and the enzyme is not consumed. The following figure depicts the intensity and absorbance at 430 nm (this is the absorption optimum for yellow) as a function of time, as recorded by the light-meter or "spectrophotometer." The notch or blip at 20 seconds corresponds to the addition of the supernatant solution. There is a region of linear absorbance change which is the time in which the reaction is proceeding the fastest. The digested plasmids were introduced into the same volume and incubated with 2X ligation buffer to allow the cut ends to re-anneal and be repaired by ligase. The products of digestion by these enzymes will have "sticky ends" - short sequences of DNA on the terminal ends of the digested fragments, which will be able to re-anneal with complementary sequences resulting from digestion by that same enzyme. Another possible recombinant is the double plasmid, formed by reannealing of one set of every fragment produced in the digest; this plasmid will not be distinguishable from the desired simple plasmid using the above-mentioned antibiotic resistance test, though the greater size of the plasmid will probably decrease the frequency at which transformation occurs, and the successful formation of this plasmid is somewhat less likely from a strictly probabilistic stance than that of the simple plasmid, due to the greater number of collision events required.
In this lab, log-phase cells were induced to become competent through incubation in calcium chloride, and the competent cells were transformed with plasmids containing genes for antibiotic resistance.
The pAMP-transformed cells grew successfully on ampicillin-containing media, indicating that successful transformation had occurred. Bacteria transformed with the ligated plasmid from the previous lab were incubated on kanamycin media overnight, resulting in successful colony growth. In a previous lab, colony cells were spun down in liquid media, lysed, and cell products purified away from the plasmid DNA and RNA. The undigested colony isolates show two bands of approximately the correct sizes to represent intact pAMP and pKAN plasmids; no extremely large plasmid DNA is evident in these lanes.
We propose a method by which a plasmid inserted into a microorganism will be used as a vehicle for encoded information.
In the meantime, incorporating equal access clauses into some licensing agreements has attracted research money from charitable organizations. Candida SchimmelCandida Albicans is een gistachtige schimmel die leeft in de darmen, het vaginale gebied en de mond.


My friend and I who worked on this project together decided to test on yeast because it is a unicellular microscopic organism which translated to English basically means that it is “weak”. We took the yeast cultures to the lab at WITS University (the lab at school does not have  equipment  advanced enough for this type of experiment) where we transferred the yeast to agar plates. Our findings does not bode well for all the people out there who thought their hands were being effectively sterilized when using the UV hand dryers for less than a minute.
No difficulties were encountered, and a suitably thin gel (~0.5 cm thickness) free of noticeable air bubbles or other inhomogeneities was produced. The use of a compromise buffer meant that no single enzyme was acting at optimal efficiency; however, all three enzymes were able to act and produce restriction digests of the genetic material. The non-transformed cells grew successfully on LB media, producing a bacterial lawn, and no colonies were observed on the ampicillin plate. Plasmid solution was incompletely thawed before use by this group, and it is possible that the thaw liquid contained no plasmid or insufficient quantities of plasmid, preventing successful transformation.
True optimal transformation efficiency is approximately (10^5) transformants per microgram plasmid. The protocol is often used with pBLUE, which induces a blue color on specially prepared plates. Increasing concentration of penicillin is additionally expected to increase the magnitude of the pH change, as a greater number of protons will be liberated in the solution. Some proportion of the resulting plasmids are expected to be recombinant, conveying resistance to both antibiotics. The re-annealed sequences can be "patched together" by DNA ligase, resulting in a new double-stranded sequence. The following diagram illustrates the desired simple plasmid (which also serves as a legend for subsequent diagrams) as well as a number of possible alternate recombination products.
Each plate contained approximately 1200 colonies (calculated as an average over several sampled sub-areas in each plate). The resulting colonies were re-streaked onto ampicillin-kanamycin media the following day, and colony growth was observed within 24 hours, indicating that some cells had been successfully transformed with genes for resistance to both antibiotics. These colonies were grown from individual cells that carried both ampicillin-resistance and kanamycin-resistance genes on inserted plasmid DNA. Digestion of plasmid DNA from both samples shows fragments corresponding to the 3755-bp "amp" fragment and the 1861-bp "kan" fragment as well as the 2332-bp fragment; the 784-bp fragment may have run off the edge of the gel. The most lucrative example of this is the recent research agreement between Jay Keasling’s lab in the Department of Chemical Engineering, the nonprofit pharmaceutical company Institute for OneWorld Health, and Amyris Biotechnologies, a for-profit biotechnology start-up. Normaal gesproken leeft deze schimmel in een gezonde balans met andere gisten en bacteriA«n in het lichaam. We reckoned that if the UV light was not effective on a single cell organism such as yeast then it would probably not be effective on bacteria and viruses that are more resilient.
When yeast is just sitting around in your cupboard it is certainly alive but is unable to bud. It is clear that for UV light to be effective one would require extensive exposure to its rays. Skateboarding and golf is on her list of sporting activities and she would love to study Forensics or Medicine (which is better than being Gwen Stefani which was her aspiration at age 3). Given a pixel size of 6.7 um square and an objective magnification of 100x, the area of the cell in pixels (2100 pixels^2) translates to a cell area of approximately 141 um. Further observatious would be expected to show a leveling growth curve, followed by a decrease as cells begin to die off. Loading dye was added to digest solutions individually, for aid in visualization during the run and to ensure that the samples would sink into the gel wells prior to running. Ethidium bromide was not added directly to the agarose in this lab; the substance is a mutagen and a suspected carcinogen, and addition of the substance to the gel prior to pouring carries a risk of aerosolization.
The blank or "control" lane shows a single fragment representing uncut phage genome, as no restriction enzymes were added to this lane. Transformation efficiency can be reduced if the bacteria are not heat-shocked properly and are thus not made competent; laboratory protocols were followed closely, however, and this possibility is less likely. It is known that transformation efficiency is 10^6 colonies per microgram of plasmid, and we can expect an average of 100 copy molecules per transformed cell.


The results of the digest were run on an electrophoretic gel to confirm the presence of digested fragments. The results of the ligation were run on an electrophoretic gel to confirm presence of appropriately sized ligation products. Incubation of transformed cells on media containing both antibiotics will suffice to kill off cells transformed with these alternate plasmids. 100 uL of transformed cells was spread on each plate (~10% of total volume), for a total of 0.005 ug of plasmid DNA spread onto each plate.
However, it is not certain whether the resistance is conveyed on a hybrid plasmid, or whether the surviving cells successfully took up copies of plasmids coding for resistances individually.
It was not known for any given colony whether the dual resistance was the result of transformation with a hybrid plasmid containing both resistance genes or transformation with one or more copies of both original single-resistance plasmids. In this lab, the plasmid DNA was digested with restriction enzyme and run on an electrophoretic gel; undigested plasmid from each sample was used for comparison of fragment length. It appears based on these results that the double antibiotic resistance observed in these colonies was due to a double transformation rather than to transformation with the large hybrid plasmid. The deal drew the interest of the Bill and Melinda Gates Foundation, which then contributed $42.6 million dollars to fund a cheaper method for producing the anti-malaria drug artiminisin.
Upon further investigation it became evident that some beauty salons and hairdressers also employ UV light to sterilize their tools. Also we figured that there was no way thirteen year olds would be allowed to test on E-coli.
We put each plate on the UV lamp according to their  allocated time and after all  that had been done we transferred the yeast that had been exposed to UV light onto potato dextrose  agar plates. Staining was instead accomplished by immersing the finished gel in a solution of ethinium promide following electrophoresis. All three are Type II restriction enzymes, which cut at a known position within the recognition sequence and thus are expected to produce digest fragments of specific lengths within a given sequence. Lane E (EcoRI) shows a distinct doublet band at the top of the lane; this is caused by the existence of digest fragments of similar size in the genome.
The plasmid-transformed cells did successfully grow and form a bacterial lawn on LB media, indicating that live cells were present after incubation.
Following transformation, we incubate 250 microliters of cell culture suspension in 25 mL LB broth; cells enter log phase growth 60 minutes after inoculation and reproduce with a period of 20 minutes thereafter. In this case, results of restriction digest were not ideal; it was speculated that incubation times were insufficient, and based on the results obtained by other groups, that a different ratio of restriction enzymes was preferable.
The re-plating step made necessary by the original error makes it impossible to calculate the original transformation efficiency, further complicating interpretation. Plasmids were isolated and digested with restriction enzyme, and fragment length was used to ascertain whether the cells contained the hybrid plasmid. Ensuring that artiminisin would be provided to developing nations at the cost of production and distribution was the key to getting that research money. We do however know that in hospitals the UV lights are kept on constantly increasing its efficacy.Considering our findings we are pretty much convinced that long term exposure would be effective as a germicide. Upon our return the yeast had flourished into thousands  of bread smelling growths all  over the plates.
As far as short-term exposure goes I doubt I will be purchasing a personal UV device anytime soon.
Over 200 minutes, the cells will exit lag phase (60 minutes) and grow through an expected seven reproductive periods; assuming the culture volume is sufficiently large to avoid slowing of growth rates over this time, we obtain an expected (2*10^5)*(2^7)=(2^8)*(10^5) cells containing a total of (2^8)*(10^7) molecules of plasmid. Also, do we absolutely have to kill all the bacteria, most of which is harmless, at the risk of weakening our immune systems?



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