DNA GESCI205 EXAM 5
Below is shown an abbreviated segment of the source DNA of the actual lac operon sequence (highlighted in grey), and the junk DNA sequences surrounding it on either side. Only one DNA strand is shown, for simplicity, but be aware that this is dsDNA and there is also a complimentary DNA strand accompanying this sequence. That image is followed by a table showing some common restriction enzymes and their restriction sites. The pDS20 plasmid map is displayed beside the table.
(To download this DNA sequence as a searchable Word document, follow this link or copy and paste it into your browser search bar: drive.google.com/u/0/uc?id=12q3MQ5_KYtLiKNs4ctGsV_EB-GCWPGO4&export=download)
Though not shown in the image of the recombinant pDS20 plasmid below, there will also be several special junk DNA sequences, known as insulators, located at various points around this plasmid. Based on what you know about how transcription of the Lac Operon works, the functions of insulators in gene expression, and the image below, in which of the following locations on this plasmid would you expect an insulator to be found? THINK CAREFULLY! (Check all that apply) NOTE: for the purposes of this question, please continue to pretend the GFP Gene is not present. I will explain why I added that here in a bit.
-Between AmpR and Ori -Between Ori and LacI -Between LacI and the Enhancer -Between the LacA and AmpR
Based on what you know about restriction enzymes and restriction sites, which of the following made-up sequences, when paired with their complimentary strand, could be a valid possible restriction site? (check all that apply)
5' - TAGCTA - 3' 5' - CGGCCG - 3'
What is a plasmid?
A circular loop of DNA used by bacteria to carry and transfer non-essential genes between each other.
After digestion with the restriction enzymes, there are three more steps you need to perform before you are finished with the gene cloning process. Questions 10 - 12 below ask you to identify these last three gene cloning steps in order. Once you have used your restriction enzyme of choice to cut the DNA, what must you do next?
Add DNA Ligase enzyme to the vial of DNA. This will fix the nicks in the plasmid backbone, effectively gluing the Lac Operon into the plasmid sequence.
What is the next and final step in the gene cloning process?
Combine the vial of DNA with a test tube containing bacteria. Then subject the bacteria to a stressful cold-hot-cold treatment to get them to take up the Lac Operon sequence. This step is called transformation.
The DNA sequence TATAAA (known as the "TATA Box") is found in virtually every bacterial promoter, and is an example of what kind of sequence?
Consensus Sequence
In nature, what do the proteins encoded by the lac operon do for a bacterial cell? (aka. what is the point of the lac operon genes? Why do bacteria have them?)
Digest the sugar lactose to provide energy for the bacterial cell when glucose is in short supply.
Below is a list of DNA sequences that play a role in the function of the Lac Operon. Which of these sequences are found within the Regulatory Region? (Check all that apply)
Enhancer Promoter Operator
9. Which restriction enzyme will you use for your experiment? Note: the pDS20 plasmid is actually a very good representation of the real cloning plasmids used by professional scientists. Unlike your homework problems, in real life there may be more than one possible choice of restriction enzyme that meets all of the requirements and is perfectly fine to use. Turns out, God didn't make every question in life a yes or no answer. Who knew? As such, although there are still several wrong answers, there is actually more than one possible correct answer to this question (and this question is keyed to recognize any of the possible right answers and mark them correct)! You are the scientist! This is your experiment! What enzyme will you use!? :D (please select your choice from the drop-down menu).
HindIII SalI XhoI
What is the general term used to describe the role that Substance X plays in the regulation of gene expression in the Lac Operon?
Inducer
What did Substance X do to the Lac Operon that made the host bacteria glow green?
It bound to the Repressor protein, causing it to let go of the regulatory region, which allowed the operon genes to be expressed.
What purpose does the AmpR gene, included on this plasmid, serve in your experiment?
It is a gene that serves as a selectable marker, encoding for the antibiotic resistance enzyme beta-lactamase.
What purpose does the pDS20 plasmid serve in your experiment? (aka. Why do you need it?)
It is a vector, needed in order to transform the lac operon sequence into the bacterial hosts for analysis.
I scrape off a few of the recombinant bacteria from your petri dish and subject them to a procedure that methylates the LacI gene on the pDS20-LacGFP plasmid (see plasmid map above). What effect do you expect this change will have on Lac Operon gene expression in these bacteria?
It will cause these bacteria to always glow green, with or without inducers being provided, because methylation of LacI will result in constant expression of the Lac Operon.
I then scrape off a few more recombinant bacteria from your original petri dish and subject them to a different procedure that methylates the Lac Operon on the pDS20-LacGFP plasmid, instead of the LacI gene. What effect do you expect this change will have on Lac Operon gene expression in these bacteria?
It will cause these bacteria to never glow green, with or without inducers being provided, because methylation of the Lac Operon will result in permanent repression of expression in the Lac Operon.
As part of the experiment, you choose to add glucose and a second substance (I'll refer to it as "Substance X" for the remainder of this section) to your petri dish of white bacteria, to see what happens. After a few minutes, your colonies start to glow green under the black light! Based on what you know about how the Lac Operon works, what is Substance X?
Lactose
To which area(s) of the Lac Operon can the Lac repressor protein bind? (Select all that apply)
Operator
After you finish doing that step, what do you do next?
Perform PCR on the contents of the vial, in order to make a bunch of copies of the recombinant plasmid.
Which of the following enzymes will we need in order to successfully clone these DNA segments into the pDS20 plasmid? Note: This is not asking for every protein that will be involved in your entire experiment, only the ones involved in the actual process of getting a DNA segment from inside one organism to the inside of another. (Check all that apply)
Restriction Enzymes DNA Ligase
Which of the following MUST be present in the DNA sequence of this plasmid in order to successfully use it in this (or any) gene cloning experiment? (Check all that apply)
Selectable Marker Restriction Sites Origin of Replication
You will notice in the image above that the LacI gene is found on this plasmid (marked by the grey box). What purpose does the LacI gene serve in your experiment on the lac operon?
The LacI gene encodes for the LacI repressor protein that participates in regulating expression in the Lac Operon, which is what you are investigating in this experiment.
These experiments with glucose and Substance X demonstrate that the lac operon is a wonderful example of combinatorial regulation! How?
They show that several different factors, including the Repressor and the Activator, all have an influence regulating gene expression in the lac operon, rather than just one factor. Different combinations of these factors acting results in different levels of expression.
E. coli bacteria, grown in optimal conditions, have the ability to go through mitosis and divide every 20 minutes, so after only a short time, a small mound of genetically identical E. coli bacteria results, which we call a "colony". Each of the milky-white spots on the petri dish is a colony of E. coli bacteria. Each colony originated as a single bacterium that was in the test tube when we spread its contents on the petri dish. Given what you know about the pDS20 plasmid and the petri dish (see paragraph at the top of the page), what useful information can you determine about the visible bacteria in these petri dish colonies?
They were successfully transformed with the plasmid.
What would happen if you were to only add Substance X to the petri dish without also adding glucose?
This would cause the bacteria to glow even brighter than before.
The Activator protein and the Repressor protein of the Lac Operon are good examples of what?
Transcription Factors
Why must this plasmid have an origin of replication (labeled "Ori" in yellow in the image above) in order for it to be useful in your gene cloning experiment?
You need an origin of replication to ensure that subsequent generations of each transformed bacteria will also contain the plasmid.
