+pGLO LB/amp/ara

 

+pGLO LB/amp

-pGLO LB

 

-pGLO LB/amp

 Photos Taken & Uploaded by: Julie Nguyen

Lesson 1: Focus Questions
Consideration 1: Can I Genetically Transform an Organism? Which Organism?
Written by: Julie Nguyen

1. A single cell organism would be better suited for the total genetic transformation because you would only need to insert the gene of interest in a single cell instead of many. There is only one cell in which contains all the DNA information needed to transform. For example, bacteria cells are single cell organisms that have an easier chance to uptake new DNA and are asexual so they can replicate at a fast rate even by themselves without the assistance of other cells.

2. An organism where the new generation develops and reproduces quickly so that you can evaluate the results faster to prevent yourself from wasting time and you can see the traits faster.If it does pass on its traits the experiment will be continued on and if results fail then scientists can confirm that faster. One that reproduces more slowly means it will take longer periods of time.

3.The organism should not produce toxins or any chemical compounds that can affect people who are doing the lab. The organism should have a fast growth rate, but should not grow or live outside of the lab and are easy to control and maintain. The organism should not infect or cause diseases to humans, animals, and plants. The organism should be easy to obtain cost wise and are accessible in the environment.  

4. A bacterium would be the best choice for genetic transformation  and being the host organism since it is a single cell organism, it is much simpler to inject the gene of interest into one cell rather than multiples. Bacteria are asexual which means they reproduce faster and new generations would be obtain quicker.They reproduce fast but not uncontrollable and safe ones do not make people sick.

Consideration 2: How Can I tell if Cells Have Been Genetically Transformed?

a) about 71 colonies

b) 1) about 1.5mm 2) about 0.5mm 3) about 1.0mm
c) White with hints of Yellow
d) evenly distributed
f) They do not have the ability to live in presence of ampicillin unless they have a gene of resistance for ampicillin. 

1. You could use a positive LB/agar plate and one negative LB/agar plates and place some ampicillin to the positive LB/agar plate to see if the E.coli cells can resist ampicillin. If they resist it they will live and continue to reproduce if not, they die.

2. I would expect that in the experimental results, the E.coli bacteria dies in the presence of ampicillin and only those with the gene of resistance to the antibiotic will live on with the colonies showing along with the gene of interest to live on while the one that stays non-competent will not have the gene of resistance to ampicillin and therefore, dies and no colonies will be seen.

Lesson 2: Review Questions
Written by: Julie Nguyen

1. You would find the +pGLO LB/amp to be like the original non-transformed E.coli colonies the most because they E.coli bacteria will grow since the LB helps provide it with nutrients and have a gene of resistance to ampicillin. However, it does not have arabinose which is the gene that transforms the bacteria to glow, +pGLO will not fluoresce and mostly be the same as the untransformed E.coli bacteria.

2. The transformed cells would most likely be located on the transformation plates (+pGLO LB/amp and + pGLO LB/amp/ara) because the genes for transformation pGLO have been added to these plates. Also, they both have the gene of resistance for ampicillin so the E.coli bacteria with pGLO will be able to survive in the presence of ampicillin.

3. The Transformation plates should be compared to the Control plates. The -pGLO plates do not have the gene of interest so they will not glow and that will let us know that the Transformation plates should glow. The +pGLO group have a gene of resistance for ampicillin so they have th most potential to be transformed but without the arabinose the cells would not fluoresce.

4. The control plate is the plate that does not have any qualitative changes to it and is used to compare it to the variables that are changed on the Transformation plates. The Control plate does not have the pGLO so it does not have any changes to it such as arabinose and ampicillin.

Lesson 3: 

B. Analysis of Results pg 44

Written by: Alexander Yiu

1) Original Trait        Analysis of Observations
     Colour                    Bacteria have a faint white colour
     Colony Size           Colony sizes are similar from before and after transformation

2) New Trait              Observed Change

Colour                    The colonies on the LB/amp/ara plate are fluorescent green under UV light

Ampicillin               The transformed colonies can grow on ampicillin resistance

3) The plasmid has to express a gene for ampicillin resistance.

4) Fluorescence and ampicillin resistance are not natural properties of E. coli. Because the E. coli have both these properties together, it is because of transformation and not just a basic mutation.

What's Glowing Page 45

1) The plasmid sample did not glow under UV light.

2) The two possible sources that can be eliminated are original bacteria and the pGLO plasmid DNA.

3) This observation indicates that the source of fluorescence is from a protein that the plasmid encodes.

4) A successful experiment will be represented by the presence of colonies on the positive pGLO LB/amp and the positive pGLO LB/amp/ara plates. That and the absence of colonies on the negative pGLO LB/amp plate. This and the fact that the colonies on the LB/amp/ara plate should shine a fluorescent green colour under UV light. An unsuccessful experiment will show an absence of colonies on the positive pGLO LB/amp/ara plates.

 Lesson 3: Review Questions Page 46

Yes the bacteria that did not receive the plasmid are growing on a plain LB plate.

 1) I can’t observe E. coli growing on the LB plate that does not contain ampicillin or arabinose because ampicillin is not present. Therefore I can’t tell if the E. coli is able to grow on the ampicillin plates. Although the transformed bacteria that have the ampicillin resistant gene should be fluorescent so I can shine a UV light on them.

2) Adding bacteria from the LB plate to the LB/amp plate will be the best test. If the bacteria are visible on the LB/amp plate then they are ampicillin resistant. If no bacteria are visible, then they were not ampicillin resistant.

3a) The two factors are arabinose and the UV light.

 

3b) pGLO is translated by the bacteria into proteins that cause they fluorescence that we see with the UV light. Arabinose controls this expression of the protein. This means that the bacteria will only create this protein when arabinose is present. When the UV light is shone, the protein will resonate and gives off the energy we see as green fluorescent light.

3c) One advantage for an organism to be able to turn on or off particular genes in response to certain conditions is that it helps the the organism to conserve energy. For example, if E. coli consumes glucose and lactose but is in a plate of glucose, it can turn off certain genes to not make lactose enzymes and waste energy.