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Vehicles for Gene Cloning

This is a discussion on Vehicles for Gene Cloning within the Biotechnology Engineering forums, part of the ENGINEERING WORLD category; A DNA molecule needs to display several features to be able to act as a vehicle for gene cloning. A ...


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Old 09-11-2008, 04:47 PM
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Default Vehicles for Gene Cloning

A DNA molecule needs to display several features to be able to act as a vehicle for gene cloning. A cloning vehicle also needs to be-
• Most importantly it must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells.
• Relatively small,
• Ideally less than 10kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate.
Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: plasmids and bacteriophage chromosomes. Besides these two, other DNA molecules are also identify to serve the purpose of cloning vehicles. The list is as follow-
1. Plasmids – (up to 10kb DNA can be cloned)
2. Bacteriophages - (up to 20-40kb DNA can be cloned)
3. Cosmid - (up to 40kb DNA can be cloned)
4. Phagemid
5. YACs - (up to 2MB)
6. BACs - ( up to 300kb DNA can be cloned)
7. Viruses as vectors.
Plasmids
Basic features of plasmids
Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell (Figure). Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium.
Marker Gene for antibiotic resistance: The ability to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence in the bacterium of a plasmid carrying antibiotic resistance genes. In the laboratory antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid (Figure).
Origin of replication: All plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell quite independently of the main bacterial chromosome also called non-integrative plasmids (Figure(a)). The smaller plasmids make use of the host cell's own DNA replicative enzymes in order to make copies of themselves, whereas some of the
larger ones carry genes that code for special enzymes that are specific for plasmid replication.
A few types of plasmid are also able to replicate by inserting themselves into the bacterial chromosome (Figure (b )). These integrative plasmids or episomes may be stably maintained in this form through numerous cell divisions, but will at some stage exist as independent elements.
Size and copy number: The size and copy number of a plasmid are particularly important as far as cloning is concerned. Plasmids range from about 1.0 kb for the smallest to over 250 kb for the largest plasmids, so only a few are useful for cloning purposes.
The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. The factors that control copy number are not well understood, but each plasmid has a characteristic value that may be as low as one (especially for the large molecules) or as many as 50 or more. Generally speaking, a useful cloning vehicle needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained.
Conjugation and compatibility
Plasmids fall into two groups: conjugative and non-conjugative.
Conjugative plasmids are characterized by the ability to promote sexual conjugation between bacterial cells (Figure), a process that can result in a conjugative plasmid spreading from one cell to all the other cells in a bacterial culture. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes, which are present on conjugative plasmids but absent from the non-conjugative type. However, a non-conjugative plasmid may, under some circumstances, be cotransferred along with a conjugative plasmid when both are present in the same cell.
Compatible and incompatible plasmids: Several different kinds of plasmid may be found in a single cell, including more than one different conjugative plasmid at anyone time. In fact, cells of E. coli have been known to contain up to seven different plasmids at once. To be able to coexist in the same cell, different plasmids must be compatible. If two plasmids are incompatible then one or the other will be quite rapidly lost from the cell. Different types of plasmid can therefore be assigned to different incompatibility groups on the basis of whether or not they can coexist, and plasmids from a single incompatibility group are often related to each other in various ways. The basis of incompatibility is not well understood, but events during plasmid replication are thought to underlie the phenomenon.
Plasmid classification / Types
The most useful classification of naturally occurring plasmids is based on the main characteristic coded by the plasmid genes. The five main types of plasmid according to this classification are as follows:
(1) Fertility or 'F' plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids, e.g. F plasmid of E. coli.
(2) Resistance or 'R' plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections, e.g. RP4, commonly found in Pseudomonas, but also occurring in many other bacteria.
(3) Col plasmids ability to synthesised colicins, proteins that kill closelt related strains that lack ‘col’ plasmid, e.g. ColE 1 of E. coli.
(4) Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, e.g. TOL of Pseudomonas putida.
(5) Virulence plasmids confer pathogenicity on the host bacterium; e.g. Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants.
Commonly used plasmids in Rec-DNA Technology are pBR322, pUC, pSP64 and pSP65, pGEM3 etc
The useful properties of pBR322
The genetic and physical map of pBR322 (Figure) gives an indication of why this plasmid has been such a popular cloning vector.
The first useful feature of pBR322 is its size. it was stated that a cloning vector ought to be less than 10 kb in size, to avoid problems such as DNA breakdown during purification. pBR322 is 4363 bp, which means that not only can the vector itself be purified with ease, but so can recombinant DNA molecules constructed with it. Even with 6 kb of additional DNA, a recombinant pBR322 molecule is still a manageable size.
The second feature of pBR322 is that, it carries two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells containing the plasmid, and each marker gene includes unique restriction sites that can be used in cloning experiments. Insertion of new DNA into pBR322 that has been restricted with Pst1, Pvu1 or ScaI inactivates the ampR gene, and insertion using any one of eight restriction endonucleases (notably BamHI and HindIII) inactivates tetracycline resistance. This great variety of restriction sites that can be used for insertional inactivation means that pBR322 can be used to clone DNA fragments with any of several kinds of sticky end.
A third advantage of pBR322 is that it has a reasonably high copy number. Generally there are about 15 molecules present in a transformed E. coli cell, but this number can be increased, up to 1000-3000, by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol (p. 44). An E. coli culture therefore provides a good yield of recombinant pBR322 molecules.
Plasmids in organisms other than bacteria
Although plasmids are widespread in bacteria they are by no means as common in other organisms. The best characterized eukaryotic plasmid is the 2um circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The discovery of the 2um plasmid was very important as it has allowed the construction of vectors for cloning genes with this very important industrial organism as the host. Other yeast plasmids so far identified are as follow-
Yip or yeast integrative plasmid
YEp or yeast episomal plasmid
YRp or yeast replicative plasmid
Yep or yeast centromere containing plasmid
All the above cited plasmids can be used as shuttle vectors which allow the gene sequences to be transferred back and forth between Yeast and E.coli. These shuttle vectors have both E.coli and Yeast replication gene. Some plasmids can retrive the gene from yeast chromosomes. These are called retriever vectors
However, the search for plasmids in other eukaryotes (e.g. filamentous fungi, plants and animals) has proved disappointing, and it is suspected that many higher organisms simply do not harbour plasmids within their cells.
Bacteriophages as vector:
Basic features of bacteriophages
Bacteriophages, or phages as they are commonly known, are viruses that specifically infect bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or occasionally ribonucleic acid RNA molecule ) carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules (Figure).
The general pattern of infection, which is the same for all types of phage, is a three-step process (Figure).
(1) The phage particle attaches to the outside of the bacterium and injects its DNA chromosome into the cell.
(2) The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on the phage chromosome.
(3) Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium.
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Old 01-18-2010, 03:54 AM
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Default Where video Avatar?

Hello everyone! Who knows where to upload the film Avatar?
I even bought the film Avatar for a SMS to ??????? ?????? | ????????? ?????? | ??????? ???? ?????? ?? rsskino.ru ?????? ?????? ????! , the link was, but download fails, the system will boot quite strange cocoa something.
Men, advise where to normal as quickly download film avatar?
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Old 03-11-2011, 09:19 AM
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Default Hello my friends

Hello, I new yours frient on this forum)
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Old 03-25-2011, 06:09 AM
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Default Windows 7

I bought Windows 7 Professional 64 Bit
Windows 7 pro. (the Windows 7 Ultimate Keygen
version for system builders NOT the upgrade) would i be able to reformat Windows 7 Enterprise Serial Key my hard drive and use the same key code or would i need to buy another one?The OEM version keys to the first system it's installed on. You can install it to the same Windows 7 Professional Activationhardware more than once, using the same key. (There are no negative effects.) You can't install it on any other motherboard, though, even if you just upgrade that computer.Can i buy a student edition windows 7 proffessional key and use that for a genuine windows 7 key since my brother has the actual DVD Cheap Windows 7himself, he actually bought the software, can i use that key for that DVD that my brother bought, which is the full version.Are you saying your brother has Windows 7 Professional Student Edition and you want to buy another license for that software? It's possible Microsoft will sell you a 2d license for Windows 7 Professional 64bit the same software, but not likely. However you can contact Microsoft directly and ask them if they will do that. But you are really just paying for the software a second time....so you may as well just go buy the software again so you can get the license (the license is the product key)No. If your Windows 7 Update Keybrother bought the full version of Windows, such as home, home premium, etc, then the only key that will work with that DVD is the same exact version of Windows. A Windows 7 student edition key will only work on Windows 7 Student Edition. The keys are specific to each version of Windows.I have seen some auctions on Ebay selling Windows 7 keys. I purchased a single-license Windows 7 Home Premium for my laptop a few months ago, and I want to Windows 7 Home Basic X64 install Windows 7 on the new desktop computer I've built. Are these numbers that are being sold legitimate and usable Windows 7 Enterprise Aktivierung Key
with my copy of Windows 7? Or Windows 7 Pro Aktivierung Key am I stuck buying the same software a second time?As a rule of thumb, don't trust anything that seems to good to be true on ebay

also, im thinking it would be a much Windows 7 Professional cl¨¦easier install if you had a disc on a home built rig, i was in this very situation last month

good news is though, you can buy Windows 7 Enterprise Serial clavewindows 7 licenses for arounf ˇę50 in some places on the net
i know its steep, but it is a very good operating system

may i recomend 7 ultimate though?
i upgraded from home premium to ultimate - very noticeable improvement

and again if youve built a mid-high spec pc - dont scrimp on the OS !
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Old 03-25-2011, 06:12 AM
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Default Windows 7

I bought Windows 7 Enterprise 32bit
Windows 7 pro. (the Windows 7 Pro 64 Bit
version for system builders NOT the upgrade) would i be able to reformat Discount Windows 7 my hard drive and use the same key code or would i need to buy another one?The OEM version keys to the first system it's installed on. You can install it to the same Windows 7 Pro 32bithardware more than once, using the same key. (There are no negative effects.) You can't install it on any other motherboard, though, even if you just upgrade that computer.Can i buy a student edition windows 7 proffessional key and use that for a genuine windows 7 key since my brother has the actual DVD Windows 7 Home Premium Keyhimself, he actually bought the software, can i use that key for that DVD that my brother bought, which is the full version.Are you saying your brother has Windows 7 Professional Student Edition and you want to buy another license for that software? It's possible Microsoft will sell you a 2d license for Windows 7 Upgrade Key the same software, but not likely. However you can contact Microsoft directly and ask them if they will do that. But you are really just paying for the software a second time....so you may as well just go buy the software again so you can get the license (the license is the product key)No. If your Windows 7 Enterprise Keygenbrother bought the full version of Windows, such as home, home premium, etc, then the only key that will work with that DVD is the same exact version of Windows. A Windows 7 student edition key will only work on Windows 7 Student Edition. The keys are specific to each version of Windows.I have seen some auctions on Ebay selling Windows 7 keys. I purchased a single-license Windows 7 Home Premium for my laptop a few months ago, and I want to Cheap Windows 7 install Windows 7 on the new desktop computer I've built. Are these numbers that are being sold legitimate and usable Windows 7 Home Basic Aktivierung
with my copy of Windows 7? Or Windows 7 Ultimate Aktivierung am I stuck buying the same software a second time?As a rule of thumb, don't trust anything that seems to good to be true on ebay

also, im thinking it would be a much Windows 7 Pro 32bitseasier install if you had a disc on a home built rig, i was in this very situation last month

good news is though, you can buy Windows 7 Pro clavewindows 7 licenses for arounf ˇę50 in some places on the net
i know its steep, but it is a very good operating system

may i recomend 7 ultimate though?
i upgraded from home premium to ultimate - very noticeable improvement

and again if youve built a mid-high spec pc - dont scrimp on the OS !
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Old 12-30-2011, 12:07 AM
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Default What is Your TOP 3 PC Games ?

Choosing amongst a myriad of brobdingnagian things was not in the least my forte, so when I had to choice my culmination ten computer games, I was definitely faced with a challenge. After some thinking (and I norm in fact opinion through), this is the heel over of games that I sensible of are worthy to be mentioned in my top ten computer games article.

Lately, tons people from playing video games on consoles such as Ps3 and Xbox 360 because of exciting games and the well-being of use. Uncountable consoles up with a advance graphics easter card that allows on account of deeper gaming experience. So does this technique the end of PC gaming era? Undeniable not. Computers gave extraction to the video engagement industry. Unlike the consoles, computers bring into the world been almost on more than 25 years. All the years of experiences unite up to haler games and it conveys the letter that pc gaming compel be here in the interest of a long time. Still today, computer gaming leads the gaming hustle because most gamers are PC gamers.

3) Let loose’s start with some Dress up Games irate birds and some green pigs.
I cannot ponder my better ten computer games list without this game. Angry Birds is a thick irregular bold, but who am I kidding? You unquestionably be sure what Angry Birds is. And if you aren’t wiped out bored with of having played it on your android phone, iPhone, tablets, iPad, roku etc, you can also play the darn fetish on your computer!

2) StarCraft
Is it lapse that more than half of my cap ten computer games consist of tactics games? The changeless cannot be said benefit of consoles. PC games differ because you in reality pull someone's leg to ‘think’ and be sharp-witted, notably in this design; Lead Craft. Individual aspects of the game such as motivation, technology, biology, and a keen-witted mystery assign this stratagem song of the most renowned physical time strategy game. This type of moving and addicting engagement cannot be ground in consoles platform. The on the other hand habit to have knowledge of what I am talking on every side is to take part in this stratagem, you are undeviating to discern then!



Computer games are the beat games in the in style gaming era. Solace games are obedient too, but absolutely not as pleasing as ones I toy with on computer in my opinion. My register of supreme ten computer games may change in the following as PC gaming exertion is leviathan and fitting for satisfied, it will mould in requital for decades to come.
[ Game Games, dress up games, Five Games for your daily fun ! Five dress up games ]
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Old 01-19-2012, 06:23 AM
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Default Breaking Point

I've always been a loser but I've never realized it until a few years ago. Never was good with people, and until very late in life I thought the only way to make friends and please people was to do and be everything they wanted us to. I shaped my personality on that of those I wanted to be friends with. I wasn't myself. So I was bullied, beaten, mocked, taken advantage of and such.

Then there were the parental units. Work took them away a lot, and the little time we spent together was them saying constantly I behaved like a lunatic and if I didn't behave properly I was to be commited to an institution. What a great thing to say to a child, right? My whole family, though small, started to see me this way. I was the crazy one, the child that never said anything that made sense, the child who always had to be watched lest he break, stole or severely damaged anything. Well, movin' on...

Let's see...School: Check; Parents/Family: Check; Work - Check my other story "damned if you do..."; That leaves married life.

I don't have to tell you about my love life, use your imagination under these circumstances. I met my future wife and eight years later we got married. Best and fastest day of my entire life. I later found out that my marriage isn't a partnership, but a domination. She's the dominating one, I'm the dominated one.

So at this point I really start thinking like that famous Demotivator poster with the picture of a sinking ship that says «It may be that your goal in life is to serve as a warning to others.». I looked around and realized that no one respected me, people saw me as a child of 5 in a 30 year-old body, the lunatic mad prankster who never took anything seriously and never did anything right. I had one last chance to prove myself to the world: to have children of my own, to make others around me see that I too could make something right, something beautiful. Then the Doctor told me I couldn't have children. I was sterile. No chance in Hell.
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