Dissection

http://youtu.be/PufiqmxGiYU

http://youtu.be/XglaL4QxEr0

Gel Electrophoresis Lab

 

In this lab, we took a sample of DNA and performed gel electrophoresis.  Gel electrophoresis is used to create restriction maps and identify DNA.  Here's what we did:

Methods:

We took 5 samples of DNA and used three separate restriction enzymes to cut it.  Each sample of DNA contained a combination of restriction enzymes, and then was placed into separate wells on the electrophoresis gel.  This is what each sample of DNA contained:

1. Marker (Only contained DNA)
2. DNA + PstI
3. DNA + PstI/HpaI
4. DNA + PstI/SspI
5. DNA + PstI/HpaI/SspI

Once each sample was placed into the gel wells, the entire gel box was placed into the gel electrophoresis machine.  Electricity is run through the gel, propelling the light (smaller) strands a DNA far away from it's original position, and propelling heavy (bigger) strands of DNA a smaller distance from it's original position.  We then tallied the size of each strand of DNA based on it's distance from the origin, and also by comparing the DNA strands to the marker in well 1.

Discussion:

By observing the positions of the strands in each well in relation to the marker, we were able to approximate the length of each strand.  When all of the strand lengths added up to 2,600 base pairs in well two, we then knew that wells three, four, and five must also have the same number of base pairs.  We were able to create restriction maps containing each restriction enzyme according to the length of DNA strands and number of base pairs in each strand.  For example, well two contained two fragments of 900 and 4700 base pairs. so therefore there must have been two restriction sites, creating two sections of DNA made of 900 and 4700 base pairs.  We were able to perform the same action on well three and four, creating restriction maps for each of their restriction enzymes, respectively.  In order to make a restriction map of DNA cut by all three enzymes, we simply superimposed each map from well two, three, and four on top of eachother.    

Here is an image of the number of base pairs per DNA strand

                                    

Here are our restriction maps for each enzyme

                                     

 

pGLO Transformation Lab

Purpose:

The purpose of this experiment is to transform E. Coli To be able to glow in the dark and become antibiotic resistant. 

Info:

The expression of certain traits is not only regulated by enzymes, but also by genes as well.  Enzymes usually regulate activity in the short term, while cells can regulate gene expression for the entire life cycle of many cells, making it long term.  Gene expression works in a away very similar to the way feedback inhibition in enzymes works.  It is controlled through regulatory genes and operons.  The regulatory gene controls the production of a certain molecule, but only with the help of an operon.  An operon consists of a promoter region, operon genes, and an operator.  The operator is a section of DNA that a repressor can bind to, and subsequently turn it off.  Inducible operons are ones that are normally turned off, but the presence of a certain substance can turn it on.  Repressible operons are ones that are normally on, and the presence of a certain substance can turn them off.  

In experiments where one type of foreign DNA is inserted into an organism, a plasmid is used.  A plasmid is circular bacterial DNA.  For example, the gene found in deep sea squid that makes them fluorescent, called GFP, is inserted into bacterial cell and creates a plasmid.  But, when scientists want to control an experiment where a plasmid is inserted into an organism ( in this case, bacteria) , another gene must be inserted into the plasmid.  In order to make sure that every bacterial cell being observed contains the gene for GFP, an gene for antibacterial resistance is also inserted into the plasmid. Because both the gene for GFP and antibacterial resistance are contained in the plasmid, it guarantees that only bacteria that contains the plasmid will survive for observing.

 

Methods:

 For the E. Coli to be able to glow in the dark we implanted the gene for fluorescence (Green Fluorescent Protein) into the bacteria via a plasmid. We used the plasmid pGLO that contains the gene for Green Fluorescent Protein as well as antibiotic resistance to the antibiotic ampicillin. We used 4 different agars all containing different combinations of various solutions (transformation solution, LB nutrient broth and pGLO) to test which would give us the end result of glow in the dark and antibiotic resistant E. Coli.

 

Discussion:

In this lab, we tested the transformation of E Coli. Our results showed that with the addition of pGLO to the bacteria, it was able to transform to become antibiotic resistant, and to glow. Our control plates show that without the pGLO, the bacteria doesn’t transform. Neither control plate glowed; the bacteria without antibiotic ampicillin grew, and the bacteria with ampicillin did not, exactly as we expected. The experimental plates with the pGLO added had different results: The plate with just pGLO, LB nutrient broth, and antibacterial insulin had bacterial growth, showing that the pGLO caused the bacteria to form antibiotic resistance. In the second experimental plate containing pGLO, LB nutrient broth, antibacterial insulin, and transformation solution, the bacteria not only grew, but also glowed. This shows that the pGLO caused the bacteria to form antibiotic resistance and the transformation solution caused the bacteria to glow. Our results are exactly as expected, with no growth in the control plates, and the different results of the experimental plates. We were expecting some variation because we forgot to let the solutions sit at room temperature for 10 minutes before putting them in the incubator. Our observations show that this didn’t seem to have an effect on the lab, as we got the same results anyway.  Our transformation efficiently was 1.9 x 10 to the 3, which shows that we had very high efficiency of the cells receiving the plasmids.  Overall, the expiriment was pretty successful.  

Conclusion:  In this experiment, recombinant DNA is was only viable (alive and glowing) when it contained the genes that coded for antibacterial resistance and glowing capabilities, and when arabinose was present.  In conclusion, plasmids may be inserted into bacterial DNA as a way of creating a recombinant cell, or, a cell that contains DNA from more than one organism.

 

Data:

 

Discussion:

In this lab, we tested the transformation of E Coli. Our results showed that with the addition of pGLO to the bacteria, it was able to transform to become antibiotic resistant, and to glow. Our control plates show that without the pGLO, the bacteria doesn’t transform. Neither control plate glowed; the bacteria without antibiotic ampicillin grew, and the bacteria with ampicillin did not, exactly as we expected. The experimental plates with the pGLO added had different results: The plate with just pGLO, LB nutrient broth, and antibacterial insulin had bacterial growth, showing that the pGLO caused the bacteria to form antibiotic resistance. In the second experimental plate containing pGLO, LB nutrient broth, antibacterial insulin, and transformation solution, the bacteria not only grew, but also glowed. This shows that the pGLO caused the bacteria to form antibiotic resistance and the transformation solution caused the bacteria to glow. Our results are exactly as expected, with no growth in the control plates, and the different results of the experimental plates. We were expecting some variation because we forgot to let the solutions sit at room temperature for 10 minutes before putting them in the incubator. Our observations show that this didn’t seem to have an effect on the lab, as we got the same results anyway.

Conclusion:

 In this experiment, recombinant DNA is was only viable (alive and glowing) when it contained the genes that coded for antibacterial resistance and glowing capabilities, and when arabinose was present.  In conclusion, plasmids may be inserted into bacterial DNA as a way of creating a recombinant cell, or, a cell that contains DNA from more than one organism.

References: Campbell Biology 9th edition

 

 

 

DNA Replication

Here's our review video about DNA replication!  DNA is arguably one of the most important processes in life, but can be a little confusing sometimes.  In this video, we broke replication down to make it easier to understand.

Watch and enjoy!

http://youtu.be/uCK2bopIeA0