transgenics and gmos- lecture 5 Flashcards
transgenic
an organism whose genome has been altered by the transfer of a gene or genes from another species or breed (sometimes called genetically modified, gmos)
when disturbed, aequrea produces green light, caused by
green fluorescent protein (gfp)
cloning
take piece of dna and park it into a bacterial cell
*take plasmid out of bacterial cell, put little fragments of jellyfish dna into the plasmid, then park the plasmid back into the bacterial cell
*persuade bacterium to express whatever is in the plasmid
*expression assay to detect successful colony, plasmid with gfp gene will glow green
plasmid
little loop of dna that can be semi independent like a second chromosome in a bacterial cell
genomic library
in eukaryotic genes, amino acid coding exons are often interrupted by non coding introns
cdna
complementary dna- converting jellyfish edited mrna into dna
creating a cdna library
need an enzyme to convert dna from rna, reverse transcription
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription.
reverse transcriptase process
1) reverse transcriptase needs a primer, and mrna has a poly a tail so primers made of thymine are used
2) reverse transcriptase leaves a small 3’ overhang, then used as a primer for dna polymerase which uses single stranded dna as a template to produce double stranded dna
3) s1 nuclease is used to cleave the bend in the dna,, leaving double stranded dna with blunt ends
to allow us to park our cnda into a plasmid, we need two enzyme tools
restriction enzyme and a ligase
expression vector
has the material necessary for expression that is not present in the gfp sequence
getting the plasmid into the bacterial cell: electroporation
electroporation causes components of the membrane to become fluid which allows things to squeeze in from the outside
how do we find the bacteria with the cdna we want
pcr cloning
a strategy for cloning a coding sequence by taking advantage of the known sequence of a cdna to simplify it via pcr before inserting this product into a plasmid
hybrid primers
part restriction enyzme recognition sequence and part gfp sequence complement
what determines when and where a gene is expressed
promoter (through interaction with regulatory molecules like activators and repressors) because where the promoter is, that is where the transcription factor binds
genetically modified organisms/transgenic organisms
any organism whose genome has been altered by the insertion of a gene or genes from other species or breeds
refers to the isolation of a DNA sequence from any species (often a gene), and its insertion into a vector for propagation, without alteration of the original DNA sequence
molecular cloning
expression cloning
In this technique, researchers isolate all of the
genes that are being expressed in an organism, and test them one by one for a function of interest. We can explore this technique by walking through its steps for identifying the gene encoding green fluorescent
protein, GFP
Reverse transcription is a process by which researchers
can make DNA copies of RNA molecules using the enzyme:
reverse transcriptase
how does reverse transcriptase start dna synthesis
To start the DNA synthesis, reverse transcriptase requires a primer, which is a short oligonucleotide that specifically binds to the RNA of interest. It is especially easy to reverse transcribe eukaryotic mRNAs,
because they all share a common sequence feature that can be used to facilitate primer design – the poly-A tail. Since every mRNA has a poly-A tail at its 3’ end, an oligo (dT) primer, a primer consisting of only thymine
residues, can be used to synthesize a complementary strand of DNA using the mRNA as the template
how do rna molecules perform a reverse transcriptase reaction
To perform an RT reaction, RNA molecules are first isolated from cells, and mixed with the oligo (dT) primers. Each primer will bind to the poly-A tail of a different mRNA, allowing the reverse transcriptase enzyme to bind the template and synthesize a strand of DNA with sequence complementary to the RNA. This generates a double-stranded RNA-DNA hybrid. In addition, the RT process adds a few additional bases to the 3’ end of the new DNA strand. This short extra piece of DNA then serves as the primer for synthesizing a second strand of DNA using the first as a
template.
To make a “complete” double-stranded DNA molecule (rather than an RNA-DNA hybrid), what needs to happen?
first the RNA is degraded by treating the hybrid
molecules with NaOH or an RNase enzyme
Then DNA polymerase I is added to the reaction. This enzyme uses the overhang left from RT as the
primer for synthesizing a second strand of DNA, forming a doublestranded DNA hairpin. To make it easier to use the DNA in future reactions, it is important to have a double-stranded DNA with free ends, not a hairpin. Therefore, after the second strand is synthesized, S1
nuclease is added to the reaction to remove the single-stranded loop that connects the complementary DNA strands. At the end of this process, you will have double-stranded DNA copies of all the mRNAs in a
cell. These are known as complementary DNAs, or cDNAs, and they can easily be purified for further use.
Since your goal is to identify the jellyfish gene that produces GFP, you can perform an RT reaction using all of the mRNAs found in the jellyfish cells expressing
GFP. This allows you to obtain cDNAs corresponding to all of the genes expressed in these cells, including the gene encoding GFP.
a collection of cDNAs representing all the mRNAs in a cell or organism is known as a:
cDNA library
expression cloning
enables you to identify your gene of interest based on its activity. Note: this technique is very useful because you can narrow down your search to only the expressed genes, as opposed to having to search the entire
genome. In this technique, each cDNA is inserted into a different bacterium (or other cell type) so that researchers can screen for the activity of the gene of interest. They do this by looking for cells that, upon
taking up a particular cDNA, begin to exhibit a specific characteristic due to their expression of the gene of interest. In this example, since you are trying to identify the gene that encodes GFP, you can look for bacteria
that turn green – they will only do so if they take up and express the GFP gene
To induce the cells to express the gene of interest, researchers will first insert a cDNA copy of the gene into a plasmid, then transfer the plasmid into the cells. A plasmid is a circular DNA molecule that is capable of directing both its own replication and the expression of the genes that it contains. To accomplish these tasks, a plasmid must contain three key features:
The first is an antibiotic resistance gene (or any gene
that allows for selection of transformed cells). This allows researchers to distinguish cells that have taken up a plasmid from those that have not. This is a key “first stage” screening tool.
The second is an origin of replication. This is a DNA sequence that will initiate replication of the
plasmid, so that, as the bacterial cells divide, all of the daughter cells will receive at least one copy of the plasmid.
The third is an MCS or multiple cloning site. This is a DNA sequence that contains convenient restriction
sites that can be recognized and cut by different restriction enzymes, allowing DNA to be inserted into the plasmid.
Molecular cloning makes extensive use of restriction enzymes to generate transgene constructs, including plasmids that contain foreign DNA inserts (such as your cDNAs). In expression cloning, you often cut your plasmid with a restriction enzyme that leaves blunt ends, which are:
both DNA strands end at the same base, leaving no single-stranded overhangs. This makes it easier to insert a cDNA from your library, which also has blunt ends, into the plasmid
After cutting the plasmid with a
restriction enzyme, the cut plasmid is mixed with the cDNA library, and what is added?
DNA ligase is added. DNA ligase connects the sugar-phosphate backbone of each plasmid DNA to the sugar-phosphate backbone of a cDNA. This generates many transgene constructs, each of which contains
a different cDNA insert
how can you transform (add) transgene constructs into bacteria
One strategy that is commonly used is electroporation – bacteria are shocked with an electric current. This temporarily causes their plasma membranes to become permeable, and each bacterium can take up
one of your plasmids. The bacteria are then plated out on media that contains antibiotics. This media is selective – only bacteria that have taken up a plasmid will contain the appropriate antibiotic resistance gene
that will allow them to survive. In this way, you can select the bacteria that have taken up plasmids, and allow them to grow. Each individual bacterium that has taken up a plasmid will form a separate colony on the
antibiotic plate. Next, bacteria that specifically took up the gene of interest can be identified because they will express the gene and so take on a new characteristic that you can “look for” in the colonies. For example, in our GFP example, you can look for bacteria that turn green as they must have taken up plasmids that contain the GFP cDNA as their insert. You can then sequence the insert to determine the sequence of the GFP gene and use it for further experiments
why PCR-based cloning instead of expression cloning
more specific, much easier, and more efficient
In PCR cloning, what do you design primers for
to specifically amplify the sequence of the gene or DNA sequence that you want to clone. Then you can use PCR to make many copies of the gene. This makes it much
easier to insert the sequence of interest into a plasmid, because you will have many copies of only your desired DNA sequence. In addition, you can add different restriction sites (the DNA sequences recognized by
different restriction enzymes) to the ends of each of your primers so that you can control the direction that the PCR product inserts into the plasmid
why is adding different restriction sites possible with pcr based cloning
This is possible because PCR-based cloning uses restriction enzymes that cut DNA in a different way from the enzymes used in expression cloning. In expression cloning, you use a single restriction enzyme to cut
your plasmid leaving blunt ends so that your cDNA insert (which itself has blunt ends) can be ligated in. In PCR-based cloning, you instead use enzymes that leave “sticky ends,” or single-stranded overhangs. Each
sticky-end cutting restriction enzyme will leave a unique overhang, or short single-stranded DNA sequence, when it cuts the DNA. This allows DNA to be inserted more efficiently (because an insert and plasmid cut by the
same enzyme will have complementary “sticky ends,” and so will find and stick to each other more easily)
In addition, you can use two different restriction enzymes that each produce unique sticky ends to control the direction in which your insert is
put into the plasmid. To do this, you add different restriction sites to the 5’ ends of each of your PCR primers, so that each end of your PCR product
will have a different restriction site on it. You can then cut the PCR product with the appropriate restriction enzymes, cut your plasmid with the same restriction enzymes, and insert your PCR product into the plasmid with the desired orientation. This is because, once cut, each end of the PCR product will “match up” specifically with the end of the plasmid that was cut with the same restriction enzyme
expression vector.
is anything that you can use to move transgenes around - plasmids are commonly used vectors in molecular biology labs, but for some purposes, researchers will use viruses or other vectors instead
Expression vectors are special because, in addition
to the antibiotic resistance gene, origin of replication, and MCS, they also have all of the sequences that you would need to express an inserted gene. You can, for example, have an expression vector that will drive the
expression of an insert at a high level in bacteria. Such a vector would need to have a strong bacterial promoter and transcription termination sequences. In addition, the gene of interest would need to be inserted into the plasmid between the promoter and terminator sequences in order to be expressed properly
microinjection
Once you have a transgenic construct that includes the promoter and gene that you want, you can generate transgenic animals that express your transgene. To do this, you should purify the transgene construct and use microinjection to introduce it into 1-celled embryos. If
you are lucky, the embryos will take up the plasmid and express the gene. And if you are really lucky, they will pass it on to their offspring!
If the transgene is incorporated into the genome of a
cell that will give rise to egg or sperm, the offspring of the transgenic fish…
will also be transgenic
reporter gene assay
The assay involves creating a construct in which we substitute a reporter gene [i.e., one that reports where it is active], such as GFP, for the actual gene. Now
the gene’s promoter is hooked up to a GFP gene. If we insert this construct into embryos, we will see GFP expression in every tissue in which
the gene is normally expressed
example of gmo food
AquaAdvantage salmon contain a transgene that causes
them to express a growth factor at a higher level than normal salmon. This is because the growth factor is being expressed under the control of a strong promoter (one that is permanently switched on, rather than, as in
the natural situation, one that is switched on seasonally). The increase in growth factor expression causes the salmon to grow much faster than
they otherwise would, allowing them to be sent to market sooner than non-GMO salmon
