Meaning of the word place. Molecular cloning, or how to place foreign genetic material into a cell
Place, - still, - eating; - yet (-yon, - ena); committed view
1. what. Determine, find a place for something Place books on a shelf.
2. whom (what). Place (in some room, housing). Place the visitor in a separate room.
3. whom (what). Give somewhere for some purpose. Place your child in kindergarten. Place him in the hospital. Place your savings in a savings bank.
4. what. Print, publish. Submit an article to a magazine. Place an ad in the newspaper.
imperfect species place, - ay, - ay.
noun. room, - I, neuter gender
Examples of using the word place in the context
- . The second one is with the upstairs neighbors, the Monastyrevs, there’s a free room now, you can place Tattoo there.
. - Well, let's tell him that I was run over by a carriage or a van and that I had to place to the infirmary.
. I would like place some things in the hotel safe.
. They had to in the end place Tom to the madhouse.
. It’s impossible, you must agree, place all my affection into the dog.
Place
Place
PLACE
I will place, you will place (you will place colloquially), owls (To put).
1. who-what. To provide a place for someone, to allow someone to settle down somewhere. Place the guest on the sofa. Accommodate tourists in a hotel.
2. who-what. Place, arrange, lay down. I placed the light source behind. All books can be placed in the closet. Place the choir at the back of the stage.
3. who-what. Identify, assign, give somewhere. (obsolete). “Old Aratov settled in the capital with the goal of placing his son at the university.” Turgenev . “My guardian uncle brought him to St. Petersburg and placed him in the service.” Turgenev .
4. What. To dispose of something, to determine the place, purpose for something. Place somewhere capital. Place your savings in a savings bank. Place somewhere orders.
5. What. Submit for publication or printing. Submit an article to a magazine. Place an ad in the newspaper.
Ushakov's Explanatory Dictionary.
D.N. Ushakov.
Contain, accommodate, squeeze in, lay down; to settle, to install, to install, to place; attach, perch, tuck in, roll up, pile up, shove in, shove in, put in, move, lay out, stuff in, dislocate, lay down, load, put in... Synonym dictionary
PLACE, estate, etc. see place. Dahl's Explanatory Dictionary. IN AND. Dahl. 1863 1866 … Dahl's Explanatory Dictionary
PLACE, eat, eat; still (yon, ena); Sovereign 1. what. Determine, find a place for something n. P. books on the shelf. 2. whom (what). Settlement (in what room, housing). P. the visitor to a separate room. 3. whom (what). Give where n. for what n.... ... Ozhegov's Explanatory Dictionary
place- place, place, place (place incorrectly) ... Dictionary of difficulties of pronunciation and stress in modern Russian language
place- what l. where and where. 1. where (to determine, arrange somewhere; give for storage, use). Place your child in kindergarten. Place money in a savings bank. [Father] took him to St. Petersburg as soon as he was eighteen years old, and... ... Control Dictionary
place- PLACE / PLACE PLACE / PLACE, arrange / arrange, arrange / arrange, unravel, owl. child, colloquial, nesov. and owls to do, talk attach/attach... Dictionary-thesaurus of synonyms of Russian speech
I owls see placed 1., 2., 3., 4. II owl. trans. see Efremova's Explanatory Dictionary. T. F. Efremova. 2000... Modern explanatory dictionary of the Russian language by Efremova
Place, place, place, place, place, place, place, place, placed, placed, placed, placed, place, place, placed, placed, placed, placed, placed, placed, placed,... ... Forms of words
Verb., St., used. compare often Morphology: I will place, you will place and place, he/she/it will place and place, we will place and place, you will place and place, they will place and place, place, place, placed, placed, placed,… … Dmitriev's Explanatory Dictionary
Books
- Cutting out snowflakes, Victoria Viktorovna Serova, Vladimir Yurievich Serov. Paper snowflakes... In whose childhood there was no this little magic, you fold a square, cut it out, unfold it, holding your breath, and here it is, a miracle, a man-made snowflake! Amazing...
Place- 1) determine a place for something (put, put, hang, arrange), 2) settle, provide housing, 3) place someone somewhere (in a hospital, in an orphanage, in a boarding school), 4 ) invest funds (money), 5) print, publish.
Examples of use: place a chair in the corner, place guests in a corner room, I was placed in the surgical department, place money in a commercial bank at interest, in the latest issue of the New World magazine for 2013 they published a selection of poems by a famous poet.
Post- 1) arrange in a certain order, 2) distribute among many persons (participants).
Examples of use: place dishes on the shelf, place linen in the closet, place orders profitably.
Fit- place something completely or in large quantities.
Examples of use: Mom was able to fit all my things on one shelf, I want to fit all the apples in one basket.
Place (s) - place (s) - fit (s)
Fit- 1) fit in, find enough space, 2) settle.
Examples of use: I didn’t think that so many people could fit here; the cereal does not fit in the jar; we stayed in a small house on the shore.
Accommodate- find a place for yourself, settle down, settle down.
Examples of use: fit in a house, in a room, in a chair, on a sofa, sit comfortably.
Fit- 1) fit completely, 2) settle down, settle in a limited space.
Examples of use: the sisters sat on one chair; I didn’t think that so many people could fit in such a small room.
Local-1) relating to the estate, 2) owning the estate.
Examples of use: local land ownership, local nobility.
Landowner- owned by a landowner.
Examples of use: manor's house, manor's estate, manor's garden, manor's stable.
Top up- increase, add, make more complete.
Examples of use: replenish your bank account, replenish your food supplies, replenish your collection.
Fill- 1) take it in its entirety, fill it out, 2) enter the required information.
Examples of use: the water was rising: it quickly filled the basements of the houses; fill out the questionnaire, form, application form.
grow old- become older or older.
Examples of use: father, grandfather, brother, matchmaker is old, mother is old, cat is old.
Be obsolete- 1) become old, 2) fall out of use, out of fashion, out of use.
Examples of use: my views are outdated, it’s time to change them; classics cannot become obsolete; research methods are outdated; the equipment is outdated.
Deed- intentional action.
Examples of use: noble act, selfless act, manly act, worthy act, perform an act.
Misdemeanor- an act that violates the rules of conduct; offense.
Examples of use: commit a misdemeanor, an unfortunate misdeed, serious punishment for a misdemeanor.
Venerable- 1) worthy of reverence, respect, 2) significant (about distance or size, volume).
Examples of use: respectable gentleman, old man; venerable goals, objectives; be at a respectable distance.
Respectful- 1) treating someone with respect or showing respect, respect, 2) significant (about distance or size, volume).
Examples of use: respectful young man, respectful appearance, respectful manners, respectful facial expression, respectful look; at a respectful distance.
Festive- 1) related to a holiday, 2) elegant, beautiful, 3) solemnly joyful, happy.
Examples of use: holiday date, holiday event, fireworks display; festive outfit, suit; holiday dress; festive look, festive mood, holiday memories.
Idle- 1) not doing anything, being idle, 2) not filled with work, business, 3) empty, useless, aimless, generated by idleness.
Examples of use: an idle and empty man, no one saw him idle; idle life, idle lifestyle, idle conversation, idle question, idle interest.
Practical- 1) related to practice, 2) involved in any business directly, personally, 3) being the application of knowledge and skills in practice.
Examples of use: practical activity, practical application, practical significance; practical guide, practical center; practical classes, practical knowledge and skills, practical techniques.
Practical - 1) knowledgeable in practical matters, successful in the practical side of life, 2) profitable, convenient.
Examples of use: practical person; practical housewife, wife, mother; practical step; practical color, material.
Provide - 1) to give the opportunity to use or own something, 2) to give the opportunity or right to do something.
Examples of use: provide opportunities, provide documents, provide freedom of choice, right; Let me decide for myself whether to give the management of the estate to a new person.
Introduce - 1) give for familiarization, 2) highlight, send as a representative, 3) apply for an award, promotion in rank, position, 4) introduce, recommend, 5) show, demonstrate, 6) portray on stage, play, 7) mentally imagine.
Examples of use: present the research results; present candidates from the region, from the school; submit for an award; introduce the groom to his parents; present prospects, direction of work; the actors successfully presented the feelings and states of their characters; imagine something, be of interest.
Representative- 1) elected, 2) reflecting the interests of all interested persons, groups, parties, 3) respectable, prominent, making a favorable impression.
Examples of use: representative power, representative authorities; representative meeting, representative congress, representative exhibition; representative man, representative appearance.
Executive - 1) for presentation purposes, 2) luxury class.
Examples of use: entertainment expenses, purposes; representative interests; executive class car, executive class (hotel) room.
Performance- 1) noun. from the verb to represent, 2) official paper, petition for an award, promotion, rank, 3) performance, theatrical action, 4) image of objects and the world in people’s perception, 5) understanding, knowledge.
Examples of use: presentation of evidence in court; presentation for an award; theatrical performance; my views, your views, get an idea of events; have a very general understanding of historical processes.
Providing- noun from the verb to provide: provision.
Examples of use: provision of living space, provision of services, provision of opportunities, provision of work in accordance with the contract.
Recognized- 1) the one who was recognized (participle from the verb. recognize), 2) appreciated, known.
Examples of use: recognized authority, recognized talent; recognized artist, actor, director, public figure, scientist.
Grateful- feeling or expressing gratitude, gratitude.
Examples of use: to be grateful, grateful words, grateful attitude.
Belittle- 1) put in a humiliating position, humiliate, 2) belittle, underestimate.
Examples of use: to belittle in one’s own eyes, to diminish the importance, to diminish the role.
Humiliate- to offend, to offend.
Examples of use: humiliate in front of everyone; humiliate with attitude, words, slap, scream.
Problematic- conjectural, unsaid, unlikely, doubtful.
Examples of use: problematic solution, statement, conclusion, assumption; problematic conclusion, result; problematic possibility.
Problem- containing a problem or intended to solve it.
Examples of use: problem situation, problem article, problem group, problem approach, problem lesson, problem lecture.
Industrial- related to or intended for production.
Examples of use: production process, production facilities, production department, industrial relations, production defects, production meeting, production territory.
Productive- producing, creating, productive.
Examples of use: productive labor, productive forces.
Prophesy- predict, foretell.
Examples of use: prophesy the future; prophesy misfortune, trouble; prophesy good luck, victory.
Say goodbye- to intend, to predict.
Examples of use: to become wives, husbands; to become a boss; to become a bride; speak for yourself, for your brother.
Fisherman- 1) one who fishes, 2) a lover of fishing.
Examples of use: Fishermen sat and stood along the banks of the lake. Passionate fisherman, amateur fisherman; a real, knowledgeable, experienced fisherman.
Fisherman- 1) one who is engaged in fishing, 2) a fishing enthusiast (colloquial)
Examples of use: fishermen worked in teams; a team of fishermen; a real, good, old fisherman.
Fishing- related to fishing or intended for fishing.
Examples of use: fishing season, fishing gear, fishing trawler, fishing fleet.
Fishing- engaged in fishing as a trade.
Examples of use: fishing artel, fishing trawler.
Vocabulary- relating to a dictionary or the work of creating dictionaries.
Examples of use: dictionary entry, vocabulary of a language, dictionary work.
Verbal-1) adjective from noun. word, 2) expressed in words, in words.
Examples of use: verbal war, battle; verbal material, verbal combinations.
Resistance- 1) resistance, 2) term: resistance of materials
Examples of use: resistance to authorities, resistance to the will of parents, electrical resistance, compression resistance, resistance to materials; windage.
Resistance- ability to resist.
Examples of use: resistance to diseases, infections, stress; body resistance; resistance of rocks to weathering.
Comparable- participle of the verb compare; one that can be compared to something.
Examples of use: comparable values, incomparable with anything.
Comparative- 1) based on comparison, 2) relative, 3) linguistic term: comparative degree, comparative adjective, comparative adverb.
Examples of use: comparative research method, comparative linguistics; comparative silence, comparative prosperity; comparative adjective, comparative degree.
Old- 1) created in ancient times, 2) ancient, old
Examples of use: antique carpet, antique coin, antique jewelry, antique books; old acquaintance, old friend.
Old-1) lived for many years, 2) old, old, 3) long in use, 4) (about time) past, 5) formerly.
Examples of use: old grandfather, old woman; old grudge, old wound, old pain, old tradition; old dress, old shoes, old house; old time, old life; old address, phone number, old data.
Glass- 1) made of glass, 2) such as glass, 3) motionless, lifeless.
Examples of use: glass glass, glassware; glass shine, glass ringing; glassy look, glassy eyes.
Glass- intended for glass or glass production, working with glass.
Examples of use: buy glass putty; glass workshop, glass factory, glass raw materials, glass industry.
Satisfying- 1) well satiating, high in calories, 2) plentiful.
Examples of use: hearty pies, a hearty dish; hearty lunch, hearty food; satisfying life, satisfying winter.
Well-fed- 1) not hungry, 2) well-fed, well-fed, 3) living in abundance.
Examples of use: a well-fed person, well-fed children, a well-fed cat, well-fed cattle; a well-fed country, a well-fed Europe.
Lucky- one who is favored by luck; successful.
Examples of use: successful entrepreneur, successful athlete; happy hunting.
Successful- 1) ending in success, good luck, 2) good, meeting the requirements.
Examples of use: successful business, successful operation; a successful film, a performance, a successful role, successful words.
Mention- words concerning someone, said not specifically, but casually.
Examples of use: mention of an actor, mention by the way, relevant mention, mention in the press.
Reminder- words for the purpose of reminding.
Examples of use: important reminders, agreement reminders, agreement reminders, self reminders, birthday reminders, computer reminders.
A huge amount of biological research begins with one simple action - foreign genetic material is introduced into a cell. This action is called molecular cloning. With its help, you can obtain genetically modified organisms, turn individual genes on and off, determine the effect of a protein on some complex process, and so on. We can say that molecular cloning is the cornerstone, the foundation, the foundation, without which many wonderful techniques would not be feasible.
However, placing “non-native” DNA into a cell is not as simple as it seems at first glance. This is a long, labor-intensive and multi-step process. Entire books are devoted to molecular cloning, and, unfortunately, in an article, even a very long one, it is impossible to cover all its subtleties and nuances - something will definitely be left out. Nevertheless, I will try to talk at least a little about what it is and what is needed to make it work.
Insert
Since we are going to insert some gene into cells, then the very first, obvious step that we need to take is to somehow obtain this gene, preferably in large quantities (since - alas! - all methods are imperfect, and we need prepare for what O Most of the copies of this gene will disappear without a trace along the way and never reach the goal).
A foreign gene introduced into a cell is called insertion genome or simply insert(see Insert). You can get it in several ways.
First, we can simply isolate it from the genome to which it belongs. Let's assume for simplicity that our insertion is some kind of elephant gene. Then we need:
- Obtain a sample of elephant tissue.
- Extract DNA from this sample (see DNA extraction).
- Isolate the gene of interest to us from this DNA and obtain it in large quantities (PCR is used for this). [Note in parentheses that obtaining a gene using PCR is possible only if we know its nucleotide sequence or at least the sequence of its beginning and end (so that primers can be synthesized). If all this is unknown to us, then we will have to first analyze the elephant genome].
Secondly, it is quite possible that the gene we need has already been isolated from the elephant genome and is present in the gene library. Then our insert can be obtained from there (in fact, we will also have to tinker with this, but less than in the first case).
And finally, thirdly, it is not necessary to use an already existing gene as an insert. If a researcher is going to work with some gene that is a figment of his imagination and does not occur in nature, then he can synthesize it artificially (see Artificial gene synthesis).
Vector
Launching a “lonely” insert into a cell, that is, the gene by itself, without any accompaniment, is a completely futile matter. There are many DNA-digesting enzymes (nucleases) floating around in the cell, which will happily pounce on our defenseless insert and cut it into pieces. As a result, the insertion will ingloriously disappear without having time to accomplish anything useful, and cloning will not lead to any results.
Therefore, to protect the insert, it is embedded in a special “vehicle” called a vector. In the most elementary case, a vector is simply a DNA sequence into which our insert is sewn and which helps it not to disappear in the cell and fulfill its purpose.
There are several types of vectors, but one of them enjoys the greatest (and deserved) love among researchers - plasmids. We'll start with them.
Plasmid
A plasmid is a fairly short and usually circular DNA molecule that floats in the cytoplasm of a bacterial cell. Plasmids are not associated with the bacterial chromosome; they can replicate independently of it; they can be “spitted out” by the bacterium into the environment or, conversely, “swallowed” from this environment. With the help of plasmids, bacteria exchange genetic information with each other, for example, transmitting resistance to some antibiotic to neighbors.
Plasmids exist naturally in bacteria. But no one can prevent a researcher from artificially synthesizing a plasmid that will have the properties he needs, sewing an insert into it and launching it into a cell. A plasmid is, one might say, a blank, a blank; Different inserts can be inserted into the same plasmid. Therefore, they try to make plasmids as universal as possible and suitable for all occasions.
In order for a plasmid to become a working vector, it must have certain important characteristics.
Reproduction
First of all, the plasmid must multiply and replicate in the cell, because otherwise it will quickly undergo degradation, and the insert gene will disappear along with it. To do this, it must contain a special sequence called the origin of replication, from which DNA doubling begins. In different species of living beings, these points have different nucleotide sequences. Therefore, if we want to create a plasmid that would replicate in two types of cells at once (for example, in both yeast and bacteria), then we need to insert two origins of replication into it.
Cutting
In addition, the plasmid DNA must have areas where it can be cut in order to insert an insert. Special enzymes called restriction enzymes are used as “scissors”. Restriction enzymes are wonderful because they cut DNA not just anywhere, but in strictly defined places, which are called restriction sites (each restriction enzyme recognizes only its own site and only cuts DNA in it - or near it). Usually, many different restriction sites located at different points are placed in the plasmid - thanks to this, it can be cut in the right place by the right restriction enzyme. A section of DNA containing several restriction sites is called polylinker(see Multiple cloning site).
Selection
The process by which a bacterium ingests a plasmid is called transformation. Under natural conditions, not the entire population of bacteria can transform at any given time, but only a part of it; cells that are capable of this are called competent(see Competence). There are laboratory methods that can be used to artificially increase the number of competent cells (some of them are described below in the chapter “How to put a vector into cells”), but still, one hundred percent competence for a bacterial culture is unattainable.
So, when adding a plasmid to bacteria, we resign ourselves in advance to the fact that O The majority of bacterial cells will remain plasmid-free and untransformed. Therefore, we will have to separate the wheat from the chaff, that is, the transformed cells from all the others. To do this, a simple but ingenious technique is used.
Let's say we have built into our plasmid a gene for resistance to some antibiotic (this gene is called selective marker, see Selectable marker). Now the cells that have “ate” the plasmid will be invulnerable to this antibiotic and will be able to live peacefully in its presence. As a result, in order to select from all the bacteria to which we added a plasmid those that were able to use this plasmid for its intended purpose, we will only need to add the appropriate antibiotic to the bacterial culture. Those cells that we need will be able to exist and divide in the presence of this antibiotic, but the rest will not be able to do this.
But this is only the beginning of selection. The fact is that when we insert an insert into a plasmid, in addition to the desired combination (insert inside the plasmid), many by-products arise (see below). Therefore, if cells are resistant to an antibiotic, this does not mean that the vector we need “sits” in them. It is quite possible that they simply ate an empty plasmid, which will be of no use to us.
Therefore, we need to carry out another stage of selection, and for this there must be another selection marker in the plasmid. This could be, for example, a gene for resistance to some other antibiotic. The subtlety here is that our restriction site will be located in the middle of this gene. If the plasmid is empty, without an insert, then this gene will be working, and resistance to the antibiotic will remain. If an insert manages to wedge itself into the middle of this gene, then the gene will be damaged, resistance to the antibiotic will be disrupted, and the growth of bacteria in its presence will be inhibited. That is, all we need to do after the first stage of selection is simply transfer our cells to medium with an antibiotic for the second selective marker and this time select those colonies that do not grow.
There are other ways to carry out selection. It is possible, for example, to place a restriction site not inside the antibiotic gene, but inside some “noticeable” gene (say, one in the presence of which bacterial cultures change color). As a result, it will be possible to distinguish the necessary colonies from the unnecessary ones simply by eye, without any manipulation. For example, the now very fashionable blue-white selection system works on this principle (see Blue white screen).
In principle, you can do without the second stage of selection, and simply analyze (for example, using PCR or electrophoresis) the plasmid composition of several bacterial clones and select the clone in which the plasmid with the insert gene “sits.”
If we are going to work only on bacteria, then the matter will be limited to all of the above. However, if our ultimate goal is to place the vector in some other eukaryotic cells, for example mammalian cells, then we have another stage of selection ahead of us.
The fact is that in most eukaryotic cells plasmids do not live long and are quickly degraded. Therefore, even if we forced the cell to “eat” the plasmid, we should not hope that our insert will now remain in this cell forever. Most likely, it will only have time to express itself a little before the vector containing it is caught by the nuclease and cut into pieces. However, if the vector was accidentally able to integrate into the genome (this event is very rare, but not incredible), then our insertion, one might say, will take root in this cell - and not only in this cell, but in all its descendants. And in order to select from all the cells those that have the vector in their genome, we will need another selective marker - a gene for resistance to some eukaryotic antibiotic (because bacterial antibiotics, as a rule, do not act on eukaryotic cells). By adding the appropriate antibiotic (for example, geneticin, see G418) to the medium in which the cells are cultured, after some time we will obtain a population of only those cells in whose genome our vector “sits”.
In addition, we can insert into our plasmid as a marker some gene with a strong individuality (say, the green fluorescent protein GFP gene). This protein glows under a fluorescence microscope, and if the cell has eaten the plasmid, it will be visible under the microscope as a glowing spot. And in plasmid-free cells no glow will be observed.
Promoters, enhancers, silencers
Before each working gene there is a short piece of DNA called a promoter. It is here that an enzyme called RNA polymerase is attached, which synthesizes RNA on a DNA template, which is the first and absolutely necessary step in gene expression. If a gene does not have a promoter, its expression cannot be started; it remains silent, and although it is present in the cell, it does not manifest itself in any way. We can say that a gene without a promoter is like a car without a gas pedal. Therefore, our plasmid must have at least one promoter region, under the control of which the insert gene can be placed.
But promoters are different.
Firstly, they differ in their strength. Some cause rapid transcription of the controlled gene, others - completely sluggish.
Secondly, prokaryotes and eukaryotes have different promoters. Prokaryotic promoters do not work in eukaryotic cells and vice versa. Therefore, it would be a terrible mistake to put a gene that should, for example, be expressed in bacterial cells, under a eukaryotic promoter - it would be the same as leaving it without a promoter at all.
Thirdly, eukaryotes have several types of RNA polymerase - they ensure the synthesis of different types of RNA. And each type of RNA polymerase recognizes only its own promoters and “does not see” others. Therefore, depending on what kind of RNA our insert encodes (for example, template or, conversely, hairpin, or maybe even ribosomal), we need to select the type of promoter that we will put in the plasmid.
And finally, fourthly, different promoters are turned on in different ways. Some are constantly active. Others are activated only under certain conditions - for example, when the ambient temperature increases or certain substances appear in the cell. In addition, in multicellular organisms in each tissue some promoters are turned on and others are turned off. It is possible, for example, to select a promoter that will be active only in neurons. Or only in the neurons of the brain. Or only in brain neurons belonging to one of the subcortical nuclei. Or only in a tiny subpopulation of brain neurons belonging to one of the subcortical nuclei. And this circle can be narrowed almost indefinitely.
And in addition to promoters, eukaryotes have other DNA regions that regulate gene expression - enhancers (which enhance transcription) and silencers (which weaken it). They, unlike promoters, may not be located close to the controlled gene, but at a considerable distance from it.
Knowing all this gives the researcher amazing freedom. Having selected a suitable promoter for the plasmid (and, if necessary, some kind of enhancer), he will be able to do almost whatever he wants with the expression of the insert gene. Well, let's say, make it highly expressed, only in muscle cells and only in response to an increase in temperature.
Protein translation
When placing a vector into a cell, a scientist may want two different things:
- So that only transcription of the insert gene occurs (that is, RNA synthesis on a DNA template - for example, this is sufficient if some non-coding RNA is introduced into the cell).
- For both transcription and translation of the insertion gene to occur (that is, expression of the protein encoded by the insertion).
In the first case, the vector is called transcriptional, in the second - expressive. Expression vectors are usually a little more complex than transcription vectors because they contain:
- Kozak consensus sequence. This long name is given to a short (about 10 nucleotides) fragment at the very beginning of the messenger RNA molecule, which, through intermediary proteins, ensures the binding of this mRNA to the ribosome (without which, as you might guess, protein synthesis is impossible). The Kozak sequence is characteristic only of eukaryotes, and it differs slightly among representatives of different species. Therefore, when creating an expression vector, it is necessary to insert into it a sequence that is characteristic of the living creature into whose cells we are going to insert the vector. In addition, the Kozak sequence can be strong and weak - that is, leading to the synthesis of a large or small amount of protein. In prokaryotes, the role of the Kozak sequence is performed by the Shine–Dalgarno sequence, which directly (in the sense - without intermediaries, unlike the Kozak sequence) connects to the ribosome, after which protein synthesis begins.
- The Kozak sequence is located before inserted gene. A after there should be several more short sections to which proteins that perform polyadenylation(see Polyadenylation) - attaching a polyadenine tail to the end of freshly synthesized RNA. This tail performs several functions, including ensuring the export of RNA into the cytoplasm and helping organize translation - that is, if we want to ensure protein synthesis based on our RNA, we cannot do without it.
- And one moment. The mRNA that serves as the template for protein synthesis can only be transcribed by type II RNA polymerase. Therefore, we need to insert into the plasmid exactly the promoter that works with this RNA polymerase.
So, we have selected all the pieces necessary for the plasmid. But it is not enough to simply connect them together - their relative position plays a huge role. For example, restriction sites must not only be numerous and diverse, but also be located in the “right” places. At the same time, we must try to ensure that the final plasmid is as compact as possible, because, firstly, it will be more stable, and secondly, it will be more readily “swallowed” by the cell. In a word, you probably already realized that the design of a good plasmid is a delicate and filigree art.
Plasmid databases
Over the few decades that molecular cloning has existed, thousands of different plasmids have been synthesized, from which giant databases have been created (for example, Addgene). These databases contain plasmids for all occasions - with different types of origins of replication, different polylinkers located in different places, different selective markers and promoters, and so on. There are those in which you can sew not just one insert, but several, and there are even those that already carry some particularly popular inserts. Therefore, as a rule, researchers do not synthesize a plasmid for cloning themselves, but buy a ready-made one. If necessary, the purchased plasmid can be “finished” by inserting or removing certain sections (and then this modified plasmid can also be added to the database). In other words, often the scientist’s task is simply to select a suitable plasmid.
Other vectors
The plasmid is an excellent vector for relatively small inserts. If the gene insert is too large, then the plasmid loses stability because its sections begin to “shuffle” with each other and get lost during replication, which is why it gradually shortens. Therefore, more stable structures are used as a vector for long inserts. For example:
- Cosmida(see Cosmid) is a hybrid of a plasmid and a phage (a virus that infects bacteria). Essentially, it is just a plasmid to which sites have been added for binding to phage coat proteins (these are called cos sites, which is how cosmids get their name). The protein shell makes the cosmid more stable, allowing longer inserts to be loaded into it.
- Artificial Chromosomes(see Human artificial chromosome, Bacterial artificial chromosome, Yeast artificial chromosome) are complex and large structures that are, in fact, microchromosomes (see Microchromosome). They are relatively stable and at the same time have a gigantic capacity; several genes can be inserted into them at once. However, their enormous size makes them much more difficult to cage.
- And finally, there is another type of vector - viral(see Viral vector). This species is so important that an entire section will be devoted to it below.
We insert the gene into the plasmid
Let's say a researcher has selected a suitable plasmid and obtained the desired insert. Now you need to connect one to the other so that you can then put it into the cells.
To do this, just take a few simple steps.
As already mentioned, the plasmid has several restriction sites - that is, areas in which it can be cut by the desired restriction enzyme. We need to select a suitable site that will be located in the place where we are going to insert the insert, and then treat the plasmid with the appropriate restriction enzyme.
After this, the insert must be processed with the same restriction enzyme, since restriction enzymes usually leave protruding ends on one of the DNA strands, and these ends must be compatible between the insert and the plasmid so that they “agree” to join. If there are no necessary restriction sites at the ends of the insert, then short DNA fragments with the necessary restriction sites at the ends can be attached to it.
And finally, we need to combine the plasmid and insert (preliminarily purified from restriction enzymes) in one test tube and add to them a special enzyme called DNA ligase (see DNA ligase), which can ligate (that is, stitch together) two DNA molecules. Of course, as a result we will receive not only the desired vector in which the plasmid is connected to an insert (let's call it a Cheshire cat with a smile), but also a whole cocktail of by-products - an empty plasmid (a cat without a smile), a closed insert (a smile without a cat), several inserts sewn together (lots of smiles) and so on. During selection, these unnecessary products will be eliminated, and only the vector will remain in our hands.
Selecting the vector
So, first we carry out selection.
- We increase the competence of the bacteria, add to them a “cocktail” obtained as a result of ligation, and then inoculate these bacteria on a medium with an antibiotic, which is our first selective marker.
- We select those bacterial clones that grow on a medium with an antibiotic - they were able to eat a plasmid with an insert, or at least just a plasmid (a cat - with or without a smile).
- We carry out the second stage of selection with these clones, depending on which gene we used as the second selective marker. For example, if this gene is resistance to another antibiotic, then we transfer the bacteria to a medium with this antibiotic and select those clones whose growth is inhibited - they managed to swallow not just a plasmid, but a plasmid into which the insert was sewn (a cat with a smile) .
- We grow the resulting bacterial culture.
And so we got it - a bacterial culture in which the vector we created lives. It is quite possible that this was our ultimate goal, and now we, calm and happy, can, for example, turn on the expression of the insert gene in bacteria and reap the harvest of proteins synthesized as a result.
But if we need a pure vector that can then be placed into other cells, then we have a problem that seems insoluble. How to rescue a vector from bacteria? After all, even if we isolate DNA from these bacteria, then in addition to the vector we will also get a completely unnecessary bacterial chromosome.
Here you can take advantage of the fact that plasmid DNA has important differences from chromosomal DNA: firstly, it is much smaller in size, and secondly, it is much more supercoiled (about what supercoil is, see DNA supercoil, or DNA supercoiling) . Therefore, it is possible to select conditions under which bacterial chromosomes will sediment while plasmids remain floating in solution. It will be enough to centrifuge the resulting sediment (so that all the bacterial DNA firmly “falls to the bottom”), and then isolate our plasmid from the supernatant (usually special columns are used for this, which greatly facilitate and speed up the work).
How to put a vector into cells
And now the desired moment has come. The researcher holds in his hand a test tube in which a clear liquid is splashing - the vector obtained with so much effort.
And then an obstacle arises in front of him. The cells into which he plans to place his vector refuse to “swallow” it.
The fact is that the lipid membrane that surrounds the cells has selective permeability - that is, it allows some particles to pass through and does not allow others to pass through. Large charged molecules (which is exactly what DNA is) cannot pass through this membrane spontaneously. And if bacteria, for example, are able to ingest plasmids from the external environment (as mentioned above), then, say, animal cells are not at all inclined to this. Therefore, in order to place a vector in a cell, the researcher has to resort to many tricks, which will be discussed now. But first, a few terms.
There are several designations for introducing a vector into a cell, depending on what vector is used and into which cells it is introduced.
AAV behaves as quietly, modestly and unobtrusively as one can expect from a virus. Almost the only thing it does once in a cell is integrate into the host genome, and almost always not in the first place that comes along, but in a strictly defined place. Judging by the currently available data, it does not cause any diseases, and therefore the immune response to it is very weak. In addition, it is capable of infecting both dividing and non-dividing cells. In a word, AAV is simply an ideal basis for a vector, although it is not without some drawbacks.
And its main drawback is its small capacity. Only very small inserts can “fit” into an AAV vector, and in this it is very inferior to, for example, lentivectors.
In addition, AAV is a defective, non-independent virus. It can only reproduce in cells that are already infected with an adenovirus (as reflected in its name). This is not a bad feature at all if we want to infect a cell culture with our vector; but if we are going to make a vector for gene therapy (methods for treating genetic - and not only - diseases, in which the body is infected with a viral vector carrying the genes necessary for this organism), then such defectiveness will greatly hinder us, because viruses will not be able to spread properly throughout the body. However, now this problem seems to have been solved, and AAV vectors have been developed that are able to reproduce on their own, without any help.
Well, let’s say a suitable virus has been selected. Now the games with his genome begin.
1. First, we need to make room in this genome - that is, throw out some genes from it. It is imperative to leave those areas to which the shell sticks (so that our vector is a full-fledged, “dressed” virus), and those genes that ensure the integration of the viral genome into the genome of the host cell (so that it can fulfill its purpose); while other areas - for example, coat protein genes - we can get rid of with a clear conscience.
2. A plasmid is made from the resulting “stub” of the genome - fragments that have already been described above are inserted (origins of replication, selective markers, and so on). In principle, such plasmids already exist in plasmid databases, and, as a rule, the researcher’s task is to select the appropriate one.
3. The necessary insert is sewn into this plasmid (with all the delights of multi-stage selection that were described above).
Now we have a small problem. Even if we place this plasmid in a cell, we will not get any viruses, because we have already thrown out (in point 1) the genes that are needed to create them. Therefore, we will have to use a little trick.
We will place not one plasmid into cells, but two. The first, main one (let's call it Pu), is the one we received in step 3. And the second, auxiliary (let's call it Me), will carry the genes that we threw out in step 1. Both plasmids will begin to multiply in the host cell. The Me plasmid will express its proteins - for example, envelope proteins and proteins necessary for the self-assembly of viruses. Since Pu has areas for the shell proteins to stick, these proteins will stick to it, and as a result we will get a virus with the necessary genes inside, which is what we wanted.
So our action plan is:
4. We select some cells that lend themselves well to transfection (this cell line is called “packaging”; usually this is the human embryonic kidney cell line HEK293, see HEK cell) and place two plasmids into them at once - Pu and Me, the main and auxiliary .
5. We wait for some time (about two days) for viruses to form. After this, we collect the environment in which the cells live - the viruses float in it.
6. We purify the resulting viruses (as a rule, centrifugation and filtration are used for this) and...
7. We use them for their intended purpose, that is, we infect the cell line on which we are going to conduct experiments.
This, of course, is only a general scheme; each specific vector has its own nuances. For example, it happens that instead of one auxiliary plasmid, two or even three are used. To create some AAV vectors, packaging cells must be infected with an adenovirus. And if we create a vector for gene therapy, which should be able to multiply in the host cell and infect its neighbors, then we will have to handle the viral genome much more carefully and clear space in it with great care so as not to disrupt the ability of viruses to reproduce independently. And so on.
Last step
So - hurray! - one way or another, we still managed to place the vector into the cells. The last step remains for us - we need to select from all the cells those that have integrated vector DNA into their genome.
Actually, for this purpose we added the last selective marker to the vector - a gene for resistance to an antibiotic that works on eukaryotic cells. We will simply constantly add this antibiotic to the environment in which our cells are located - as a result, only those that have this gene in their genome and all our vector DNA in addition will survive and be able to divide.
All! Cloning is complete. We have obtained a line of genetically modified cells in whose genome our insert is present. The time has come to carry out the necessary experiments with these cells.