IDC - 4U5 Biotechnology at Dunbarton H.S.

Bacterial Transformation Using pGLO and Analyzing Temperature Reduction on Transformation Efficiency

IDC-4U5

For: Mr. Melegos

16/01/2012

Authored By: Michael Wright, Meena Sharif, Ashley Young, Alanna Takashima

Bacterial Transformation Using pGLO and Analyzing Temperature Reduction on Transformation Efficiency

IDC-4U5

Abstract

This lab works to develop the understanding of bacterial transformation through the integration of a plasmid into E-coli bacteria. Understanding of plasmids, GFP, expression of genes and bacterial candidates is used to formulate a lab which demonstrates a variety of factors associated with transformation efficiency. It was deduced that there are certain requirements present in the pGLO plasmid for full gene expression and that a reduction in water bath temperature for the heat shock portion of the lab has a negative effect on transformation efficiency.

Introduction

(To hear a narrated version of the information simply select the "Click Here" icon at the end of each paragraph and then select "Download" in the top right corner of the new window)

The pGLO lab that was undertaken is wholly reliant on the concept of bacterial transformation. Bacterial transformation is the genetic alteration of a cell based upon the uptake of foreign DNA. Upon the introduction of a gene, in this case pGLO, the genetic information is absorbed into the plasmid of the bacterium. It is then coded for as if it were a part of the original DNA.₍₁₎ This new DNA is classified as recombinant DNA. There are two different methods of initiating bacterial transformation. The first method is that of electroporation. By administering an electric shock to the cells, the membrane becomes permeable to foreign DNA.₍₃₎ Once the cell membrane is permeable the pGLO plasmid can be introduced and uptaken by the bacteria quite easily. The second method of bacterial transformation is a combination of a calcium chloride solution being introduced followed by a heat shock. The calcium chloride solution's negative charge ensures that the DNA phosphates which have a positive charge are not broken down during the heat shock process. The heat shock is then used to increase the permeability of the membranes present in the bacteria which allows for the easy introduction of the foreign DNA. Both methods work on the principle of allowing for an external DNA source to enter the bacteria and begin coding with the rest of its DNA. The first experiment which pertained to the theory of bacterial transformation was undertaken by Frederick Griffith in 1928.₍₁₀₎ Through his understanding of the relationship between different strands of a pneumococcus bacteria he was able to develop a pathogenic mixture that was extremely fatal. By combining a non-pathogenic strand with a pathogenic strand that was heat treated a mixture was developed that contained no virulent bacteria yet killed all of the subjects it was tested upon. The observations recorded in the completion of this lab led to the development of entire fields of study that are present in today's society under the broad title of biotechnology. (click here).

The bacteria modified in this lab is a strand of the E-coli bacterial family. This is an effective bacterium to use in an effort to demonstrate bacterial transformation because of the many characteristics that the bacteria possesses. The first factor that lends itself to the use of E-coli is its incredible replication rate. In as little as twenty four hours a single bacteria can multiply to form visible colonies. This fast rate of reproduction allows for results to be observed in a timely manner. Another factor that supports the use of E-coli is the way in which their cell membrane is formulated. Through the introduction of a calcium chloride solution and heat the membrane becomes porous and allows DNA to enter the DNA. As there is no defined nucleus in the bacteria the foreign DNA has direct access to the bacteria's DNA. This easy access lends itself well to the process of creating recombinant DNA.₍₂₎ The final factor of E-coli that makes it so effective in this lab is the construction of its genome. The DNA that codes for the bacteria's characteristics is quiet well understood and is able to accept the pGLO plasmid effectively. Based on all of these characteristics it becomes evident that E-coli is the most effective candidate for transformation in this instance.₍₉₎ (click here).

The success of the this lab also hinges on the presence of plasmids of DNA in the E-coli. A plasmid is an arrangement of DNA that takes a circular shape.₍₇₎ Originally evolved from bacteria, plasmids are autonomously replicating and express many of the genes responsible for antibiotic resistance. Plasmids can also be modified to express proteins and consequently other characteristics. In this lab the pGLO plasmid is introduced to E-coli DNA to create the characteristics that are desirable in the host bacterium. Contained within the pGLO plasmid are three gene sets that code for different characteristics.₍₄₎ The first of these genes is a Beta Lactamase gene. The presence of this gene in the DNA forces the bacterium to code for Beta Lactamase which renders the subject resistant to ampicillin. The second gene is the GFP gene. Extracted from Aequorea victori, a bioluminescent jellyfish, the Green Fluorescent Protein causes the E-coli colonies to glow in the presence of arabinose. The final component of the pGLO plasmid is the araC regulator protein.₍₂₎ This gene causes a protein to be made that regulates or controls transcription of the GFP. Each part of the pGLO plasmid plays a role in the success of the lab. Each part is important and creates the variation in the samples that this lab is based around. (click here).

The hook associated with this lab is the ability to make bacterial colonies glow. This glowing effect is caused by a gene extracted from the Aequorea victori jellyfish. These jellyfish are found in the Pacific Ocean off the coast of Vancouver and California. Little is known about why the jellyfish demonstrate bioluminescence or how it became a genetic trait. The leading theory pertaining to the use of bioluminescence by the jellyfish is a form of communication between individual organisms. A theory on the subject of the physiological function of GFP is in its role as an electron donor. When agitated from an outside source the GFP can act in a role similar to chlorophyll during photosynthesis and transfers electrons to another compound. When considering the physiological role of GFP there is another factor in which it acts as an important player. GFP turns the blue luminescence created by aequorin, another protein present in the jellyfish, into green fluorescent light. Despite the indiscernible role that the presence of chemiluminescence plays in the life of the jellyfish, GFP occupies many important roles in the developed world. These include many applications in the field of medicine and science. By inserting GFP into subjects that need to be tracked scientists are able to more easily identify processes in the body that were hidden due to a lack of indicators. GFP acts as an indicating dye allowing doctors to track the flow of various compounds within the body and allows for more accurate diagnostic decisions.₍₅₎ (click here).

Although GFP provides the most impressive observations for the lab there are multiple components to the pGLO plasmid including the Ara operon. The Arabinose operon is responsible for regulating the expression of the GFP in the bacterial colonies. An operon works on a principle such that if one component is not present in the environment of the bacteria than certain genes will not be expressed. In the case of the Ara operon if there is no arabinose in the environment in which the bacteria is found then the GFP will not be expressed. In the cases when arabinose is present the effector will be present to trigger a RNA reaction.₍₈₎ This reaction will allow for the expression of the GFP and under UV light the bacterial colonies will glow with a green light. By including the Ara operon in the pGLO plasmid the expression of genes can be analyzed and a connection to the bacteria's environment can be deduced. (click here).

The final component of the pGLO plasmid is a gene that gives the E-coli bacteria resistance to the antibiotic ampicillin. Ampicillin is an antibiotic that has been used to treat a myriad of different ailments which the human population faces. Closely related to penicillin, Ampicillin shares many of the same physical characteristics as well as the ability to work at effectively killing bacteria.₍₆₎ Classified as a semi-synthetic derivation of penicillin, ampicillin is most effect when used as an oral medication. It is effective at killing a variety of different bacteria including E-coli and Haemophilus influenzae while also being used as a general treatment to aid in the fight against such diseases as salmonellosis, Listeria meningitis and urinary tract infections. Overall it is used as a wide spectrum oral treatment for a myriad of common ailments as it is effect at removing bacteria, it is fairly non-discriminatory and the risks associated with its ingestion are minimal. (click here).

The original objective of this lab is to identify the transformation efficiency E-Coli under various conditions and trying to identify the expression of the pGLO gene that was inserted into half of the samples. As this lab was modified above and beyond its original parameters there were other factors set to identify. The secondary objective that is present in the lab is the identification of which factors have positive and negative effects on the transformation efficiency of the E-coli bacteria. By having each group of students perform the lab while changing one variable a data pool will be created which allows for the clear identification of which factors effect transformation efficiency. The four factors being changed are the presence of calcium chloride solution, the length of the heat shock, the increase in temperature of the heat shock and the decrease in temperature of the heat shock. The anticipated result for this lab is the decrease in transformation efficiency as the heat shock temperature is reduced by ten degrees Celsius. In relation to the initial lab, the presence of ampicillin should kill the bacteria in the sample which does not contain the pGLO gene whereas the glowing green colour will only be present in the environment containing both pGLO and arabinose. (click here).

Methodology

Prior to the completion of the lab and the recording of results there was a process of advanced preparations that made the entire lab run smoothly. The first step in the advanced preparation was the production of the nutrient agar gel which would provide the bacteria with the nutrients it needed in its initial growth stage. The agar was prepared by adding 500mL of distilled water and an LB/Agar nutrient mixture together in a one litre Erlenmeyer flask. The contents of the flask were swirled to mix the contents and boiled to remove any possible contaminants in the gel. As the solution begins to cool forty different plates were labelled. Eight plates were labelled as "LB" and were used to contain the starter E-coli colonies, eight plates were labelled "LB" for use by the four groups in the lab itself, sixteen plates were labelled "LB/amp" for use by the four groups in the lab, and the final eight plates were labelled "LB/amp/ara". Once the labelling was completed the solution contained in the flask was deposited in all of the plates labelled "LB". Each plate was filled to approximately half of its total volume. Ampicillin was then rehydrated from a dried state through the addition of 3mL of transformation solution. This newly rehydrated antibiotic was added into the agar/LB broth solution and swirled to mix. The new solution containing both LB broth and ampicillin was then distributed to the plates labelled "LB/amp". Upon the completion of filling the plates arabinose was also rehydrated. By adding 3mL of transformation solution to the dried arabinose it was rehydrated and promptly placed in the remaining solution found in the flask. This new solution now contained LB broth, ampicillin, and arabinose. The final eight plates, those labelled "LB/amp/ara" were filled with the solution. All the plates were stacked together based upon the contents of the gel they possessed and left to solidify for forty eight hours.

The plates were made approximately one week before the lab was conducted but the starter plates were filled with E-coli only one day before the rest of the lab would take place. The E-coli was provided in a dehydrated form and needed to be rehydrated before it could be added to the LB plates. A sterilized pipette was used to transfer 250µL into the vial containing the lyophilized E-coli. Upon the insertion of the solution the new mixture is left to stand for five minutes. At the end of the five minute time the vial was shaken to distribute E-coli evenly throughout the solution. A sterile inoculating loop was then inserted into the vial and proceeded to streak one of the LB plates that was used as a starter plate. A new loop was used and the process was repeated until all eight of the starter plates were properly prepared. The plates were then stored in an incubator overnight at 37^oC for use the next day. The pGLO plasmid was also rehydrated one day before the lab was conducted by adding 250µL to the lyophilized pGLO plasmid DNA. Once the transformation solution was fully incorporated the entire solution was stored in the fridge for distribution on the day of the lab.

The success of any lab lies in the organizational techniques that are adopted. By staying organized throughout the completion of the lab everything runs smoother and results are more precise. Based on this the first step that was taking was the labelling of four micro test tubes. Two test tubes were labelled "+pGLO" and the other two were labelled "-pGLO". Once the tubes were labelled 250µL of transformation solution (CaCl₂) was inserted into each tube and they were placed on ice. A micropipette was then inserted into the stock plasmid DNA tube and some of the solution was placed into both of the "+pGLO" tubes. All of the tubes were then placed on ice for ten minutes. As the tubes are sitting on ice the various plates that were prepared earlier were distributed and labelled. Two of the "LB" plates were labelled with "-pGLO" and so too were two "LB/amp" plates. "+pGLO" was then marked on two "LB/amp" plates and two "LB/amp/ara" plates. Once the ten minutes had elapsed for the cooling of the four sample tubes there was a heat shock. One set of tubes, a "+pGLO" and a "-pGLO", were placed in a foam rack and inserted into a water bath with a temperature of 42^oC for fifty seconds. Simultaneously the other set of tubes was inserted into a water bath at 32^oC for fifty seconds. This difference in temperature was the factor that the group was responsible for testing which was not commonly found in the original lab outline. This heat bath is responsible for increasing the permeability of the cell membrane of the E-coli and making the uptake of the pGLO DNA more effective. By changing the water bath temperature the group expected a reduction in transformation efficiency. Upon the completion of the fifty seconds both sets of tubes were moved back onto ice for a duration of two minutes. At the conclusion of the two minutes the tubes were removed from the rack and placed on the work bench where 250µL of LB broth was added to each tube using sterile pipettes. The tubes were than incubated at room temperature for ten minutes. Each tube was flicked repeatedly to properly mix the contents held within. Once mixed a sterile pipette was used to distribute 100µL from the various tubes into their appropriate plates. Samples were taken from the "-pGLO" sample that was treated to the 42⁰C water bath and placed on a "LB" plate and a "LB/amp" plate. The "+pGLO" tube that was treated at the same temperature water bath had samples inserted into a "LB/amp" plate and a "LB/amp/ara" plate. The distribution of samples was being done in the same divisions for the samples that were treated to the 32^oC water bath. A sterile pipette was used to distribute each sample. Once each sample was deposited on a plate a sterile loop was used to spread the sample on the plate allowing for separated colonies to form. The conclusion of day one of the lab was placing the lids back onto each of the plates and taping them together for incubation at 37^oC overnight.

After two days had elapsed the bacteria was removed from incubation and observations were recorded. The number of colonies present in each plate were compared as was the demonstration of the glowing characteristic which the pGLO gene was supposed to instill in the bacteria. The difference in transformation efficiency was also analyzed between the separate data sets in the group and between groups.

Click Here for a link to a video demonstrating the methodology and the final results.

Results

Please refer to the Appendix; Figure 1, for a table of observations.

The table effectively demonstrates all of the expected results that were established at the outset of the lab. The transformation efficiency was negatively effected by the reduction in water bath temperature as is proven by the visible results found in the table. The comparative bacterial growth on each plate also follows specific trends. The plates that contained only LB broth and E-coli had a substantial amount more bacteria than any of the other plates. Once ampicillin was added to the plates with -pGLO samples all growth was eliminated because there is a lack of resistance naturally present in the bacteria. Those with the pGLO plasmid in their DNA were able to survive the presence of ampicillin as there is a gene present which creates a resistance to it. This is demonstrated in the table as the second highest bacterial populations were found in the LB/amp plates for the +pGLO samples. The only subjects that glowed green were the +pGLO samples that were in the presence of arabinose. The table works to demonstrate that the expected results have data to support them as correct.

Please refer to the Appendix; Figure 2, for a table of transformation efficiency calculations.

Discussion

This lab was chock full of observations and different outcomes however the expected results which were stated at the beginning of this undertaking were all proven to be true. The plate containing the -pGLO sample and LB broth went on to create the greatest amount of bacterial colonies in both tests. This is due to the fact that the bacteria faces no difficulty in reproduction. The only thing present on the LB plate was food for the bacteria therefore their reproduction was fast and efficient. The strain of E-coli being used for the lab has no resistance to ampicillin. In regular cases ampicillin kills E-coli and this was demonstrated in both tests. The -pGLO samples which were placed in the "LB/amp" plates rendered no bacterial growth at all. The ampicillin did its job and killed the bacteria. As the bacteria was killed upon its deposition on the plate there was no way for it to reproduce and therefore no colonies formed. One of the genes associated with the pGLO plasmid codes for a resistance to ampicillin. Based on this the fact that some of the E-coli which was introduced to the pGLO plasmid survived on a plate of LB broth and ampicillin is quiet logical. There is not the same amount of unrestricted growth present as there was in the -pGLO and LB plate because not all of the E-coli bacteria uptake the pGLO DNA. As such some of the E-coli survive but not in the same numbers as those not exposed to any antibiotics. The other component of the pGLO gene is the ara operon. The result of having this gene present in the DNA is the restriction of expression of the GFP. The GFP will not demonstrate its characteristic green glow unless there is arabinose present in the environment. This explains why the plates containing the +pGLO samples and arabinose glow under UV light whereas the plates containing +pGLO and no arabinose do not.

The results of this lab also work to demonstrate that there are many factors present which effect the transformation efficiency of E-coli and the pGLO plasmid. One group chose to remove the transformation solution from the lab procedure and found a severe reduction in their transformation efficiency. This proves that calcium chloride is paramount in the success of attaining bacterial transformation when applying the methods used in this lab. Another group increased the water bath time by 100% and found that this increase in time led to a minor reduction in transformation efficiency. By increasing the time in which the samples are in contact with heat transformation efficiency is reduced slightly however not in the drastic manner which was present upon the removal of the transformation solution. The third group raised the temperature of the water bath by 10^oC. This also caused a reduction in the transformation efficiency of the E-coli. Finally this group reduced the temperature of the water bath by 10^oC. Like expected transformation efficiency was reduced as a result of the drop of water temperature. These results work to prove that many of the factors present in the lab including the time and temperature of the water bath as well as the use of transformation solution have been optimized to yield the greatest transformation efficiency possible.

The transformation efficiency of our bacteria is substantially lower in the reduced water bath portion of the lab however all the values that were interpolated for transformation efficiency were lower than average. This group recorded the lowest transformation efficiency in the class and the numbers that were derived were below the expected average. This was due to the method in which bacteria were taken from the starter plate. Other groups in the class swiped the plate and took multiple colonies of E-coli to start their labs with. As per instructions this group selected one colony from the starter plate to begin the lab with. As there was less genetic material present on this group's plates at the beginning of the lab the lower transformation efficiency is expected. When compared as a ratio to the approximate number of colonies the other groups started with the numbers are much closer.

As this lab is so dependent on precision and effective movement there are many sources of error that could affect the final results. The first source of error is the time it takes to transfer between the various temperature ranges. Specific times are set out to yield the most effective transformation efficiency however because the transfers are reliant on human movement from place to place there is a delay. This delay limits the effectiveness of the temperature change and limits the efficiency with which the lab is performed. Another source of error is the water bath itself. Although the water bath can be set to specific temperatures upon the introduction of the tubes which had been resting on ice the overall temperature of the water drops for a small amount of time. As it has been proven that a reduction in temperature has an effect on the transformation efficiency the reduction in temperature caused by the cold tubes being inserted into the water are a viable source of error. To fix this the water should be raised to a few degrees above the desired temperature and then lowered incrementally down to the desired temperature to remove the possibility that the water becomes too cold. A final source of error is the precision of much of the equipment that was used. As the water baths and incubator had number settings and not degree settings the temperature had to be deduced using a thermometer. As the thermometer needs to be in an environment for a fair amount of time and is sensitive to heat from human contact the exact temperature values were not achieved. As such these slight variations in temperature due to the lack of precise instruments had the potential to change the results and may explain the lower transformation efficiency which this group experienced.

The complex nature of this lab lends itself well to extensions to be created to delve deeper into the theories the lab sets out to demonstrate. One such extension is a comparison lab in which the length of the water bath is reduced. As it has been proven that an increase in the length of the water bath has a detrimental effect on the transformation efficiency a lab could be designed to see if a reduction in time has the same effect. Instead of doubling the water bath time a lab could halve the water bath time and analyze the effects. A second lab extension is the use of lesser differences in temperature difference. It has been proven that comparatively large temperature differences have a negative effect on the transformation efficiency of the bacteria however if temperature changes were applied in less drastic circumstances then the effects could be analyzed. By reducing and raising the water temperature by 5^oC a threshold could be identified as to when transformation efficiency begins to drastically reduce.

As bacteria occupy many facets of everyday life there are many ethical implications associated with bacterial transformation. The variation present in bacteria is a splendid facet of life as bacteria have many useful applications in the real world. Despite this they can also be very dangerous. This lab has proven that bacterial transformation can lead to bacteria becoming resistant to antibiotics. If strands of bacteria are developed that are resistant to antibiotics or through the use of antibiotics strands are isolated and reproduced for genetic qualities which lead to a resistance "super bugs" can be created and cause widespread death and illness. With the knowledge that bacteria can be dangerous those that endeavour to develop new bacterial strands or bacteria with transgenic DNA must be aware of the possibility of fatal consequences. In contrast by developing bacteria that are helpful such as those found in yogurt and cheeses a more efficient process can be developed for the formulation of those products. The variety of bacteria present on earth leads to a variety of applications, both good and bad, for them to occupy. Based on this the human population has to be aware of what they are doing when changing something as the consequences could be extremely negative and irreversible.

References

1. BACTERIAL TRANSFORMATION:LABORATORY EXPERIMENT - Biology Teaching Thesis. (n.d.). Georgetown University: Web hosting. Retrieved January 16, 2012, from http://www8.georgetown.edu/centers/cndls/applications/postertool/index.cfm?fuseaction=poster.display&posterID=3024

2. BIORAD. (2000). pGLO Bacterial Transformation. Biotechnology Explorer, 1, 1 - 66.

3. Bacterial Transformation. (n.d.). Plattsburgh State Faculty and Research Web Sites. Retrieved January 16, 2012, from http://faculty.plattsburgh.edu/donald.slish/transformation.html

4. Green Fluorescent Protein - The GFP Site. (n.d.). Green Fluorescent Protein - The GFP Site. Retrieved January 16, 2012, from http://gfp.conncoll.edu/

5. Jellyfish Protein GFP in Biomedical Research: Nobel Prize for developing a New Way to Study Biological Processes | Suite101.com. (n.d.). John Blatchford | Suite101.com. Retrieved January 16, 2012, from http://john-blatchford.suite101.com/jellyfish-protein-gfp-in-biomedical-research-a73309

6. New encyclopedia brittanica, the. (1983). London: Britannica Books.

7. Semple, A., & Avano, D. (2006). Nelson biology (2nd ed.). South Melbourne, Vic.: Thomson Nelson.

8. Sharwood, J., & Gordon, J. (2008). Nelson chemistry. South Melbourne, Vic.: Thomson Nelson.

9. Teachers Guide: Bacterial Transformation. (n.d.). General Atomics Sciences Education Foundation. Retrieved January 16, 2012, from http://www.sci-ed-ga.org/modules/dna/bactrans.html

10. The First demonstration of bacterial transformation. (n.d.). University of Illinois at Chicago - UIC. Retrieved January 16, 2012, from http://www.uic.edu/classes/bios/bios100/summer2003/freddy.htm

Appendix

Figure 1: Observation Tables

A: 42^oC Water Bath Bacteria Plates

Control Observations Table (42^oC Water Bath)
Diagram		Colony Count	Comparative Bacterial Growth	Physical Appearance
Control Plates	-pGLO ; LB	536	Most (1st)	- Milky White colour - Slight Yellow Tinge - Many miniscule colonies - Colonies are connected to form a smear effect - No glowing under UV light
Control Plates	-pGLO ; LB/amp	0	Least (4th)	- No growth
Transformation Plates	+pGLO ; LB/amp	102	More (2nd)	- Milky white colour - Large amount of well separated colonies - No glowing under UV light
Transformation Plates	+pGLO ; LB/amp/ara	31	Less (3rd)	- Milky white colour - Slight Yellow Tinge - Well separated colonies - Glows green in presence of UV light.

B: 32^oC Water Bath Bacteria Plates

Control Observations Table (32^oC Water Bath)
Diagram		Colony Count	Comparative Bacterial Growth	Physical Appearance
Control Plates	-pGLO ; LB	407	Most (1st)	- Milky White colour - Slight Yellow Tinge - Many miniscule colonies - Colonies are connected to form a smear effect - No glowing under UV light
Control Plates	-pGLO ; LB/amp	0	Least (4th)	- No growth
Transformation Plates	+pGLO ; LB/amp	15	More (2nd)	- Milky white colour - Large amount of well separated colonies - No glowing under UV light
Transformation Plates	+pGLO ; LB/amp/ara	4	Less (3rd)	- Milky white colour - Slight Yellow Tinge - Well separated colonies - Glows green in presence of UV light.

Figure 2: Table of Transformation Information Calculations

A: 42^oC Water Bath Bacteria Plates

Total Number of Green Fluorescent Cells	Total Amount of pGLO DNA		Fraction of Plasmid DNA on Plate
31 cells	(DNA µg)=(concentration of DNA)(volume of DNA) = 0.08µg/µL x 15µL = 1.2µg		Fraction of DNA Used = Volume spread on LB/Amp plate Total sample volume = 100µL 510µL = 0.196078431 = 0.20
Micrograms of pGLO DNA		Transformation Efficiency
pGLO DNA spread in µg = (Total amount of DNA)(Fraction of DNA) = (1.2µg)(0.20) = 0.24µg		Transformation Efficiency = Total Number of Cells __ pGLO DNA spread on plates (µg) = 31 Cells 0.24µg = 129 Transformants/µg = 1.29 x 10² Transformants/µg

B: 32^oC Water Bath Bacteria Plates

Total Number of Green Fluorescent Cells	Total Amount of pGLO DNA		Fraction of Plasmid DNA on Plate
4 cells	(DNA µg)=(concentration of DNA)(volume of DNA) = 0.08µg/µL x 15µL = 1.2µg		Fraction of DNA Used = Volume spread on LB/Amp plate Total sample volume = 100µL 510µL = 0.196078431 = 0.20
Micrograms of pGLO DNA		Transformation Efficiency
pGLO DNA spread in µg = (Total amount of DNA)(Fraction of DNA) = (1.2µg)(0.20) = 0.24µg		Transformation Efficiency = Total Number of Cells __ pGLO DNA spread on plates (µg) = 4 Cells 0.24µg = 17 Transformants/µg = 1.7 x 10 Transformants/µg

IDC - 4U5 Biotechnology at Dunbarton H.S.

Monday, 23 January 2012

IDC - 4U5 ISU

Friday, 20 January 2012

pGLO Lab Methodology and Results Video

Tuesday, 17 January 2012

pGLO Manuscript - Side Note

pGLO Manuscript