pglo Worksheet Wednesday, January 31, 2018

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1 pglo Worksheet Wednesday, January 31, :57 PM pglo Worksheet final 2 Page 1

2 An organism composed of a single cell is better suited for genetic transformation--it can transform at a faster rate - we will get more accurate results An organism that reproduces more slowly is better for passing on traits to offspring and future generations --more data is passed, hence why it occurs faster - more specific That it doesn't penetrate and enter another organism's system easily. We want it to be harder to transmit to other living organisms around - so it won't affect our ecosystem, the environment, or the one doing the lab A bacterium would be the best host organism. Bacteria are small, single-celled organisms which reproduce quickly and easily - this makes it optimal for an experiment on genetic transformation. Also it is also relatively safe and a bit less dangerous pglo Worksheet final 2 Page 2

3 Equal amounts of cells could will be placed on two different LB nutrient agar plates. One will contain just LB nutrient agar and another will contain the LB nutrient agar ampicillin. We will compare the growth of the E-Coli on both plates. If no or very little growth - negatively affected by ampicillin. If transformation rate higher and more successful- ampicillin has little effect and Ecoli is resistant Without the nutrient - the cells won't transform and grow successfully With nutrient - more resistant to Ampicillin and will grow at a faster rate pglo Worksheet final 2 Page 3

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10 On the LB/AMP/ plate, because the ampicillin will be working and will slow down/stop cell growth. It has no nutrient broth to aid the cells to grow. Doesn't give it necessary supplements pglo Worksheet final 2 Page 10

11 On the LB/AMP/ plate, because the ampicillin will be working and will slow down/stop cell growth. It has no nutrient broth to aid the cells to grow. Doesn't give it necessary supplements On the LB/AMP/ARA plate because it is the plate where the nutrient broth will provide the cells with what they need to be able to transform and grow. The LB/AMP won't experience growth and transformation at a quick rate because the ampicillin will work and slow down cell growth The +DNA LB/AMP/ARA and the +DNA LB/AMP. It will show us whether the nutrient broth will work or not. The control plates allow us to see the difference that the nutrient broth and the Ampicillin play. It will also help us determine the difference in effectiveness between +DNA and -DNA. pglo Worksheet final 2 Page 11

12 LB/AMP/ARA: lots of bacterial growth LB/AMP: very little bacterial growth occurred - still a little bit the rest have no bacterial growth Green LB/AMP/ARA: 486 LB/AMP: 6 - however they aren't glowing green under UV light blank pglo Worksheet final 2 Page 12

13 stopped by ampicillin Not glowing green The antibiotic ampicillin would do its job and stop cell growth The bacteria (E-Coli), wasn't glowing green before Resistant to ampicillin Glowing green in UV light After inserting the nutrient broth, the E-Coli became more resistant to the effects Of Ampcillin After the experiment, the plate with the nutrient broth (LB/AMP/ARA) was glowing green in UV light, something that the other plates and E-Coli before couldn't do They can also possibly become resistant to the antibiotic ampicillin and be able to transform and grow Without the Nutrient broth, the plate +DNA LB/AMP was transforming at a slow rate. However, the plate containing the nutrient broth (+DNA LB/AMP/ARA) was able to produce E-Coli cells at a much higher rate. This means that by adding the nutrient broth, we gave E Coli cells what they needed to be more resistant to ampicillin and grow pglo Worksheet final 2 Page 13

14 When we shined the UV light onto the negative pglo plasmid DNA we did not see anything significant happen and the colonies were unaffected. When we shined the UV light on the positive pglo plasmid DNA we saw the colonies in the plates fluoresce a green colour. This shows that protein is present in the plate and it fluorescing due to it These sources include the pglo plasmid DNA and the original bacterial cells The source of fluorescence is from the plasmid. The pglo plasmid has a green fluorescent protein that allows the transformed E.coli to glow under UV light. A somewhat successful experiment was represented by the presence of colonies within the (+) LB/amp/ara plate. The (+) LB/amp/ara plate also fluoresced green under the presence of UV light. On the other hand, the (-) LB/amp had no sign of colonies, which further proved the success of the experiment. One specific factor of the experiment that did not help the success of the experiment was the presence of colonies among the (-) LB/Amp plate and the glowing of the colonies under the UV light. The (+) LB/Amp plate showed no sign of colonies. There may have been an error within the transfer of loops that may have caused the success rate of this experiment to lower. pglo Worksheet final 2 Page 14

15 Yes - there is a tiny bit It is not possible to tell if the bacteria are ampicillin resistant by looking at them on the LB plate. This is because both types of bacteria, those which are ampicillin resistant and those that are not, look very similar to each other. Fill it with ampicillin to see if they will be killed by the antibiotic. A factor inside the plate that has to be in the bacteria s environment for one to observe the expression of the GFP gene is the sugar arabinose in the agarose plate. Another factor that must be present in the bacteria s environment includes the UV light, allowing the GFP protein within the bacteria to emit a fluorescent glow. The sugar arabinose turns on the expression of the green fluorescent protein by binding to a regulatory protein that is sitting on the promoter region. When arabinose is present it binds to regulatory protein, therefore changing its shape, facilitating the transcription of the gene by RNA Polymerase. The advantage for an organism to be able to turn on or off particular genes in response to certain conditions would allow the gene to adapt in different conditions and would be able to prevent overproducing of unnecessary proteins pglo Worksheet final 2 Page 15

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17 486.8 ug pglo Worksheet final 2 Page 17

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19 486 colonies 10 ug 48.6 pglo Worksheet final 2 Page 19

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