Plasmids

Characteristics involving plasmids

  1. extrachromosomal spherical Geonomics molecules which are not the main bacterial genome

  2. size range: 1-200 kilobytes

  3.  take functions advantageous to the host such as:

       i. produce enzymes which break down anti-biotics or chemical toxins

       ii. produce limitation and adjusting nutrients

   4.Duplication is coupled to host replication in a:

        i.stringent way - one (or two) plasmids produced during every circular associated with microbial duplication

        ii.relaxed method - 10-200 copies of the plasmid made during each round of bacterial duplication; (this can be increased to 1000-2000 plasmids by stopping host protein synthesis and replication with the antibiotic chloramphenicol)

UnknownDNA is inserted into a plasmid DNA (or any cloning vector) by ligating the DNA into a complementary site in the plasmid DNA. These sites are generated by digesting the DNA and vector with the same restriction enzyme. (The site for the restriction enzyme that is chosen should only be represented once in the plasmid DNA. Therefore, when the plasmid is digested, a single, linear molecule would be generated.) The foreign DNA is then inserted into the plasmid by the action of the enzyme DNA ligase. The next step is to insert the ligated DNA into a bacterial cell for propagation. This is done by a technique called transformation. Bacterial cells tend to be given either Ca2Cl or Rb2Cl. This treatment generates pores in the bacterial cell wall and membrane through which the plasmid DNA enters. Although there is no size limitation to the ligation reaction, transformation efficiency is dictated by the size of the plasmid.

Impact of plasmid size on transformation effectiveness

Molecule size (kb)	%  Maximum probability
2.0	57
3.2	100
4.3	86
12.5	43
20.0	36
39.0	14
54.0	6

The goal of the ligation reaction is to insert the foreign Geonomics into the vector. An undesirable ligation product also occurs - religated vector. One way to minimize this event is to treat the vector with phosphatase. This removes the terminal phosphate group from the restriction site of the vector and theoretically prevents religation of these two ends. In reality though, this treatment is never 100%, so a low-level associated with religation will occur. Therefore following transformation you will have two types of cells: those which contain the original plasmid and those that contain a plasmid DNA containing foreign DNA.

Plasmids are designed to distinguish the two types of transformation products. pBR322, the initial widely used vector, utilizes differential gear anti-biotic screening to tell apart the two types of transformation products. Let's say that we clone into the BamHI site of the vector. The insert Geonomics will then split the gene responsible for tetracycline resistance. But at the same time frame, the gene for ampicillin resistance is left undamaged. Transformed cells are first grown on bacterial plates containing ampicillin. This will kill all the cells that do not contain a plasmid. But we still cannot say which cells contain foreign DNA. Those cells that grew on ampicillin are then replica plated on plates with ampicillin and tetracycline. Individualstissue which grow in the presence of the Principen, but die under tetracycline selection contain plasmids which have foreign DNA inserts.

pBR322 would be a discovery for molecular biology, but the double screening procedure was time consuming and could be subject to error. In1981, a new number of plasmids were developed that permitted the identification of the foreign DNA containing cells in a single screening step. Theseare known as the pUC plasmids. As with pBR322, ampicillin resistance is used as one selectable marker. The actualsecond marker is based on insertional deactivationfrom the E. coli lacZ gene. The wild type gene can hydrolyze a specific dye [X-Gal (5-bromo-4-indoyl-B-D- galactopyranoside)] to a blue colour, and the bacterial colony is stained blue. A multiple cloning site has been put in to this gene. Thissite will accept fragments ending in a number of different restriction enzymes. Onattachment of DNAinto this site, the exerciseof the gene is eliminated and the colony appears white in color. Thereforetransformed colonies that contains plasmids with inserts can be distinguished from those with plasmids without inserts based on the color of the colony and the capability of the colony to develop on an ampicillin containing media.

cDNA Cloning (Cloning EukaryoticmRNA)

cDNA cloning isa technique for gettinga DNAcopy of the mRNAs that are expressed at a specific stage in the development of the plant. Inthis method, you can improvethe library that you will screen for those sequences withinwhich you are fascinated. The reagent requiredfor this type of cloning approach is mRNA. These mRNAs have a poly A+ tail. This buttpermits the isolation of poly A+ mRNA by using eitheroligo-dT or oligo-U columns. Total RNA is run through one of these columns under conditionswhich favor the binding of the tail to the matrix on the column. After thecolumn is extensively washed, the conditions are usuallychanged and the bound mRNA is isolated. This is the starting reagent for cDNA cloning.

Steps in cDNA Cloning

1.     Bind oligo-dT to the actualpoly +A tailof the mRNA.
2.     Add reverse transcriptase and make a Geonomicscopy of mRNA (cDNA). Erase the mRNA with alkaliand highheat. (First strandsynthesis)
3.     Add a C-tail to the 3' end of the cDNA with terminaltransferase.
4.     Add oligo-dG to the tailed-cDNA and make the actualsecond strand with reverse transcriptaseor the Klenow fragment of DNA Polymerase I.
5.     Add dC's to the 3' end of the double-stranded cDNA with terminaltransferase
6.     Add C-tailed, ds-cDNA to G-tailed, PstI cut pBR322 and anneal. (DNA ligase is not neededfor this step.)
7.     Transform E. coli cells.
8. ChooseTetr/Amps cellular material. Most, howevernot all inserts maybe removedby PstI digestions.