Purpose: The purpose of this lab was to learn about genetic transformation by moving genes from one organism

to another with the aid of a plasmid. The objectives were to perform a genetic transformation as well as determine the degree of success in our efforts to genetically alter an organism. Our hypothesis was; if a plasmid carrying the gene that codes for Green Florescent Protein (GFP) undergoes transformation into E. coli bacterial cells, then the E. Coli cells will obtain the trait. To determine this we used differently prepared plates and hypothesized the plate containing the +pGLO LB/amp will grow bacteria that does not exhibit the GFP gene, the plate containing the +pGLO LB/amp/ara will grow bacteria that does exhibit the GFP gene, the plate containing the pGLO LB/amp will not grow any bacteria, and the plate containing the pGLO LB will grow bacteria that does not exhibit the GFP gene. pGLO incorporates a special gene regulation system, which can be used to control the expression of the fluorescent protein in transformed cells, as well as codes for a resistance gene to the antibiotic ampicillin. This GFP gene can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium. The transformed cells will appear white on the plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient medium.

Methodology: Transformation Procedure: Two closed micro test tubes were labeled +pGLO and pGLO, then placed in the foam test tube rack. Next, the test tubes were opened and, using a sterile transfer pipet, 250l of transformation solution (CaCl ) was transferred into each tube. The tubes were then placed on ice. Using a sterile loop, a single colony of bacteria was picked up from the starter plate and transferred to the +pGLO tube, where it was immersed into the transformation solution at the bottom of the tube. The loop was spun between the index finger and thumb until the entire colony was dispersed in the transformation solution (with no foaming chunks). The tube was placed back in the tube rack on ice. Using a new sterile loop, this same process was repeated for the pGLO tube. The pGLO DNA solution was then examined with the UV lamp and observations were noted. A sterile loop was immersed into the pGLO plasmid DNA stock tube and withdrawn, then mixed into the cell suspension of the +pGLO tube. The tube was then closed and returned to the rack on ice. The pGLO tube was also closed but plasmid DNA was not added to the tube. The tubes were left on ice for 10 minutes. While the tubes were icing, four LB nutrient agar plates were labeled on the bottom as follows: 1) LB/amp plate = +pGLO, LB/amp/ara plate = +pGLO, LB/amp = -pGLO, LB = -pGLO. Using the foam rack holder, both the (+)pGLO and (-)pGLO tubes were transferred into a water bath at 42C for exactly 50 seconds. After 50 seconds both tubes were iced again for 2 more minutes, then were removed and placed on the tabletop. Both tubes were opened and, using a sterile pipet for each, 250l of LB nutrient broth was added to the tube just before it was reclosed. This was repeated with a new sterile pipet for the other tube. The tubes were then incubated at room temperature for 10 minutes. The closed tubes were tapped with fingers for mixing to occur. Using a new sterile pipet for each tube, 100l of the transformation and control suspensions was placed onto the appropriate nutrient agar plates. Using a new sterile loop for each plate, the suspensions were evenly spread around the surface of the LB

nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. The plates were then stacked together and placed upside down in the 37C incubator overnight. In this lab a gene resistant to antibiotic ampicillin was introduced into a bacterial strain that is killed by ampicillin. If the susceptible bacteria were to incorporate the foreign DNA, they would become ampicillin resistant. To detect this the plasmid also had the gene for fluorescence (GFP) that would be exhibited in the +pGLO LB/amp/ara plate. Selective media was used for the growth of only selected microorganisms. For example, if a micro organism is resistant to a certain antibiotic, such as ampicillin, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance gene, from growing. In this lab, it was used to prevent the E Coli. cells that did not possess the plasmid (the resistance gene), from growing and to show that the cells transformed with the plasmid (resistance gene) did exhibit resistance to ampicillin and were able to grow in its presence. Each different plate was used for separate purposes. The pGLO LB/amp and pGLO LB plates were used as control plates. Both of the pGLO plates did not have the plasmid transferred into its DNA so they neither exhibited the fluorescence gene nor were resistant to the antibiotic ampicillin. The first pGLO plate with LB/amp was used because no bacterial growth should have appeared on that plate. The second plate (-pGLO LB) was used to make sure that the bacteria did grow across the plate because this plate did not have the ampicillin medium therefore the regular E coli cells should have grown completely across the plate. The +pGLO plates were used as transformation plates because if the experiments went correctly, both plates should have grown colonies on the ampicillin medium due to their resistance. The +pGLO LB/amp plate was used to see if the cells actually inhibited the ampicillin resistance and GFP genes. The +pGLO LB/amp/ara plate should have also grown the bacteria but should have also shown fluorescent green under the UV light. The E coli cells were used because E coli is the most common bacterium in the human gut. It is able to reproduce very rapidly and a single microscopic cell can divide to form a visible colony with millions of cells overnight. It also has no nuclear envelope surrounding the bacterial chromosome and thus no true nucleus, so all of the genes required for basic survival and reproduction are found in the single chromosome. Some E coli cells also contain plasmids, small DNA molecules that carry genes for certain functions. E coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. These cells are said to be competent . Cells are made competent by a process that uses calcium chloride and heat shock. Cells that are undergoing very rapid growth are more competent more easily than cells in other stages of growth. The cells used were in the log phase of E coli cell divisions, where division is rapid.

The statistical techniques we used included:

Transformation efficiency= Total number of cells growing on the agar plate Amount of DNA spread on the agar plate (in g) -This was used to give an indication of how effective the lab was in getting DNA molecules into bacterial cells. This number represents the total number of bacterial cells that expressed the green protein divided by the amount of DNA used in the experiment, which tells the total number of bacterial cells transformed by one microgram of DNA. (DNA in g) = (concentration of DNA in g/l) X (volume of DNA in l) -This formula determined the total amount of pGLO plasmid DNA used in the experiment. In this experiment, 10 l of pGLO were used at a concentration of 0.08g/l. Fraction of DNA used = Volume spread on LB/amp plate (l) Total sample of volume in test tube (l) -This was used in determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate. This was necessary because not all the DNA added to the bacterial cells was transferred to the agar plate. 100 l of cells containing DNA were spread from a test tube containing a total volume of 510l of solution. pGLO DNA spread in g = (Total number of DNA used in g) X (fraction of DNA used) - This was used to determine how many micrograms of pGLO DNA were spread on the LB/amp/ara plates.

Results:

Table 1: +pGLO LB/amp

Observations -Many transformed colonies of bacteria (about 75) -Colonies appear white

+pGLO LB/amp/ara

-Many transformed colonies of bacteria (about 75/80) -Appear white when exposed to room light but fluoresce bright green when exposed to UV light

-pGLO LB/amp

-No bacterial growth present on this plate

-pGLO LB

-An even lawn of bacteria is present on this plate. This lawn appears off-white

The results in Table 1 showed that there was cell growth on the +pGLO LB/amp, +pGLO LB/amp/ara, and pGLO LB plates and there was no growth on the pGLO LB/amp plate. The plates that did show bacterial growth showed the bacteria to be whitish. The two +pGLO plates exhibited about 75 colonies and the pGLO LB plate exhibited a complete coverage of the plate.

The transformation efficiency =

190 0.16 g Transformation efficiency= 1187 transformants/g This means that the total number of cells growing on the agar plate was 90, and the amount of DNA spread on the agar plate was 0.16g. Discussion: The hypothesis being tested was; if a plasmid carrying the gene that codes for Green Florescent Protein (GFP) undergoes transformation into E. coli bacterial cells, then the E. Coli cells will obtain the trait. We expected the plate containing the +pGLO LB/amp will grow bacteria that does not exhibit the GFP gene, the plate containing the +pGLO LB/amp/ara will grow bacteria that will exhibit the GFP gene, the plate containing the pGLO LB/amp will not grow any bacteria, and the plate containing the pGLO LB will grow bacteria that will not exhibit the GFP gene. Our results confirmed this hypothesis and these projections. These results were able to be predicted because if the experiment was performed correctly, the ampicillin resistance gene would have inhibited the cells of both +pGLO plates which would have allowed the E coli cells to grow on the ampicillin medium. The cells would also have been able to grow on the pGLO LB because although the cells did not have the ampicillin resistance gene, they were not placed in the ampicillin medium. The pGLO LB/amp plate did not exhibit any bacterial growth because the E coli cells placed on that plate were not transformed and therefore did not have the ampicillin resistance gene which caused them to die when placed on the ampicillin medium. The cells grown on the +pGLO LB/amp/ara plate not only grew but they also exhibited the GFP gene which caused the cells to glow fluoresce green when placed under the UV light. This glowing is a result of the addition of the plasmid that contained the GFP gene. These results are congruent with those of other groups which leads us to be confident in our results. Another factor which increased the confidence of our results was that the predictions we had initially made based off of genetic reasoning were correct. The experiment was also conducted with very few flaws and a small room for error because new sterile equipment was used in each transfer of liquid which eliminates any possible chance of cross contamination. All of our equipment was working properly and everything was timed to the second. We are very confident in our results. This experiment relates to other experiments that involves transforming cells with genes that are resistant or that exhibit a specific gene. This uptake of foreign DNA alters the recipient DNA which is like Griffith s experience with harmless/harmful pneumococcal cells. This experiment was very well constructed and easy to follow. The lab could have been improved if we were told prior to the start of the lab to take notes on our observations of the original plate of E coli bacteria so that there was something to compare the new data to. This lab could be applied to the finding of cured for diseases such as cancer and ADIS because knowing that we are able to alter the genes and genetic composition of cells means that we should be able to alter and manipulate the genes found inside disease ridden cells to get rid of the infected areas. This would be incredibly useful in the finding of cures in the near future because it is easy to test the effectiveness of newly transformed cells and it would be a new approach to the finding of a cure.