Laboratory 5 Report Results

Name 4

Laboratory 5 Report

  1. Results

GFP Ligation

M-GFP ligation

LB plate

Lawn

some white, some slight green

Lawn

white

LB/Kan plate

7 Green, 10 white

70 colonies : ALL white

Table 1: Observations and results of transformation experiments: GFP ligation. LB plate contains permissive medium that allows the growth of all organisms. LB/Kan plate restricts any growth of cells that are not resistant to the antibiotic kanamycin.

1 2 3 45 6 7

Figure 1: The labeled gel photo of my restriction enzyme analysis of plasmid DNA. The first lane in the left runs undigested plasmid vector. It is followed by undigested GFP plasmid A prep and GFP plasmid B prep in the second and third lane, respectively. The fourth lane represents plasmid vector with Mlul site digested. The fifth and sixth lanes contain GFP plasmid A with Mlul site digested and GFP plasmid B with the gene, respectively. The final lane contains 1kb ladder for future analysis.

Table 2.1: Table reporting observations of the transformation experiment plates.

Table2.2: Excel Worksheet that shows variance and standard deviation in the serial dilution experiment. The number below Data (-5), (-6), and (-7) are calculated by converting the colony counts provided in table 2.1 into the number of cell/ml of the original sample. The specific formula used for the analysis is: (number of colonies on the plate)/ (volume of the plate in ml * dilution factor). Dilution factor for -5, -6, -7 are 10-5, 10-6, 10-7, respectively.

  1. We obtained lawns of bacteria in the LB plates for both GFP as shown in Table 1. Ligation and M-GFP ensured that the plate was permissive, which allowed the growth of E-Coli bacteria. Some bacteria on LB plate for GFP ligation exhibited greenish color under the UV light, which suggests that some of them experienced the transmission of plasmids with GFP gene. However, the presence of colorless bacteria on LB plate for GFP and mutated GFP ligation experiment does not indicate whether or not those microorganisms contain any forms of plasmids. This is because bacteria without plasmids can also grow on the agar plate since there is no antibiotic present. On LB/Kan plate for GFP ligation (the bottom left box of the table 1), there were seven green bacteria colonies and ten white colonies. This indicates that plasmids with the GFP gene got transformed into bacteria. It also confirms that plasmids without GFP gene underwent transformation in the GFP ligation experiment. Finally, only white colonies survived in LB/Kan plate for M-GFP ligation. This indicates that transformation also took place in the plate for M-GFP ligation. The number of bacteria that underwent transformation was confirmed by looking at the results on the restrictive plates. This is because the bacterium cannot grow in the presence of kanamycin, irrespective of whether it has GFP gene or not, unless it contains the plasmid. Thus the number of surviving colonies was less on the LB/Kan compared to LB plate since not all bacteria underwent transformation.

(2) The digested plasmid travels less than the undigested one asshown in Figure 1. This indicates that the plasmid with Mlul site islarger in size. Based on the 1kb DNA ladder on the final lane, weestimated that the size of the original undigested plasmid vectorfragment is about 2.5 kb and that of the digest one is around 3.5 kb.The size of the undigested GFP plasmid A fragment was about 3.0 andit travelled less than the regular one. This was attributed to thepresence of the extra GFP gene. The undigested GFP plasmids A and Bon the second and the third path travelled further than the digestedones on the fifth and the sixth lanes due to their larger size.

(3) The Table 2.2 contains data that are derived from the number ofbacteria counted in Table 2.1. The values contained in Table 2.2shows the number of cells per ml based on their dilution factors.According to the results of the serial dilution experiment shown inTable 2.2, the number of colonies for all (-5), (-6), and (-7) of mygroup (#5) are bigger than the calculated mean of the combined dataprovided by the class. In the case of (-5), our group obtained376,000,000 colonies while the average numbercalculated by the class as a whole was 335,454,545. Similarly, ourgroup got 450,000,000 and 300,000,000 colonies for (-6) and (-7),respectively, while the mean values for the class were 410,909,091and 290,909,091. The dilution factor increased as the sample got morediluted. Similarly, the dilution factor rose as the standarddeviation of the number of bacteria colonies increased.

(1) Based on the 1kb DNA ladder, we conjectured that our group’splasmid A contained the GFP gene. It was given that the size of theplasmid is 3.5 kb and the size of GFP gene is 1kb. Our digestedplasmid A had the size of 4.5 kb. It matched with the estimated sizeof the plasmid and GFP gene combined. The group concluded thatplasmid A is more likely to contain GFP gene. This is because thesize of the digested plasmid B did not match the estimated size. Theuncut plasmid DNA samples ran further on the gel due to the nature ofsupercoiling. The circular bacteria DNA naturally undergoessupercoiling, turning into a more condensed form. It is smaller thanthe size of the linear DNA. It is then cut by the restriction enzyme,which cannot supercoil. Errors might occur due to lack of inserts.This can result in the failure of the plasmid to bind with theinsert. In addition, an error can occur when skipping the mixing stepbecause the inserts are less likely to be in the ideal position forligation in the absence of proper integration.

(2) The serial dilution experiment is performed in order to estimatethe number of the original bacteria. Thus, the idea result would havesimilar numbers for all (-5), (-6), and (-7). To obtain a moreaccurate estimate, we can take the mean of the average values. Wefurther diluted the sample in order to facilitate the countingprocess. By taking the error estimate across (-5), (-6), and (-7)data, the deviation estimate between (-5) and (-6) can be calculatedas follows: (410,909,091-335,454,545)/335,454,545, which is around 15%. This value is not significantlyhigh. The error estimate between (-6), and (-7) data is also notconsiderably big. Thus, our estimation for the number of bacteriacalculated from the class data is useful in terms of statistics. Themost reliable data for the number of competent cells/ml would be theone for (-5). This is because the data has the smallest standarddeviation. It means that all groups gathered data closer to theaverage value. The most likely sources of error would be the simplemanual mistakes that might have occurred when counting the number ofcolonies. In addition, the number can be varied according to thesanitation level in which the experiment is performed since the datais highly sensitive to such environmental condition.