P bluescript
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The document discusses pbluescript, a hybrid vector derived from plasmids and phages, designed for producing single-stranded DNA and RNA transcripts. It details its notable features, such as multiple cloning sites, RNA polymerase promoters, and its utility for molecular cloning techniques including Gateway and TA cloning. Additionally, it covers the GUS reporter system used for quantifying gene expression through visual detection methods.
P bluescript
- 1. pBLUESCRIPT
- 2. pBluescript is an example of a combination between plasmids and phages (phagemids). Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA. Features 1. Like pUC vectors, which pBluescript is derived from, there is a multiple cloning site inserted into the LacZ' gene 2. pBluescript also contains the origin of replication of the single-stranded phage f1, which is related to M13 phage vectors. This translates into that a cell harboring a recombinant phagemid, if infected by f1 helper phage that supplies the single-stranded phage DNA replication components, it will produce and package single-stranded phagemid DNA.
- 4. 3. Produce RNA transcripts pBS has a T3 promoter on one end side and a T7 promoter on the other terminal end. This is key because it enables one to isolate the double- stranded phagemid DNA and transcribe it in vitro with either of these two phage polymerases to produce pure RNA transcripts to coincide with either of the two different strands
- 5. • 2961 basepair plasmid • Derived By Replacing pUC19 polylinker of pBS (+/-) with synthetic polylinker • SK designation indicates that the polylinker is oriented such that the lacZ transcription proceeds from Sac I to Kpn I. • 21 unique restriction sites in the multiple cloning region • Blue/White color selection • In vitro RNA transcription with T3 or T7 RNA polymerase •Double and Single-stranded sequencing Summary-pBluescript
- 6. pBluescript II SK, either the + or - orientation. The + and - denote which single strand of DNA, either the sense or anti-sense strand, can be rescued using a helper phage. These + or - orientations occur in the f1 region of the plasmid. Thus: f1 (+) origin: (consists of 3-459 base pairs) f1 filamentous phage origin of replication that allows for the recovery of the SENSE strand of the lacZ gene f1 (-) origin: (consists of 3-459 base pairs) f1 filamentous phage origin of replication allowing recovery of the ANTISENSE strand
- 7. • Extensive set of restriction sites in polylinker, many with unique 4-base overhangs • Blue/white section • Opposing T7promoters for making RNA transcripts in either direction or double-stranded RNA • Single-stranded (M13) DNA replication origin. • Compatible with pUC/M13 sequencing primers pLITMUS 28i and 38i
- 8. GATEWAY Cloning Technology The GATEWAY Cloning Technology is based on the site-specific recombination system used by phage l to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination sites called attP in phage l site and attB in E. coli. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage l encoded protein Int (Integrase) and the E. coli protein IHF (Integration Host Factor). Upon integration, the recombination between attB (25 nt) and attP (243 nt) sites generate attL (100 nt) and attR (168 nt) sites that flank the integrated phage l DNA The process is reversible and the excision is again catalyzed Int and IHF in combination with the phage l protein Xis. The attL and attR sites surrounding the inserted phage DNA recombine site-specifically during the excision event to reform the attP site in phage l and the attB site in the E. coli chromosome.
- 10. The GATEWAY reactions are in vitro versions of the integration and excision reactions. To make the reactions directional two slightly different and specific site were developed, att1 and att2 for each recombination site. These sites react very specifically with each other. For instance in the BP Reaction attB1 only reacts with attP1 resulting in attL1 and attR1, and attB2 only with attP2 giving attL2 and attR2. The reverse reaction (LR Reaction) shows the same specificity.
- 12. Step 1 Cloning the gene of interest into an Entry Vector using the BP Reaction. Step 2 Subcloning the gene of interest from the Entry Clone (Step 1) into a Destination Vector using the LR Reaction producing the Expression Clone. The ultimate goal of the GATEWAY reactions is to make an expression clone. This is often a two step process:
- 13. The gene of interest is cloned into an Entry Vector and flanked by the attL1 and attL2 recombination sites. The Entry Vector is transcriptionally silent and contains the gene for kanamycin resistance (Kmr). To produce the Expression Clone the gene has to be subcloned into a Destination Vector that contains all the sequence information necessary for expression • the gene for ampicillin resistance (Apr), • and two recombination sites (attR1 and attR2) that flank a • gene for negative selection ccdB (the encoded protein is toxic for the standard E. coli strains).
- 14. The two plasmids are mixed and the LR CLONASE Enzyme Mix is added. The reaction is directional and specific, so that attL1 only reacts with attR1 and attL2 with attR2. The recombination yields two constructs: the intended Expression Clone and a by-product (Donor Vector). The produced expression clone is under two forms of selection: the antibiotic resistance and the negative selection by the toxic ccdB protein. High levels of positive clones (typicaly more than 99%) are obtained after transformation to a standard cloning or expression strain like DH5a or BL21
- 15. One of the main advantages of the GATEWAY Cloning Technology is that once you have made an Entry Clone the gene of interest can be easily subcloned into a wide variety of Destination Vectors using the LR Reaction
- 16. TA Cloning exploits the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. This polymerase lacks 3' to 5' proofreading activity adds a single, 3'-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanines at the 5' end as this maximizes probability of adding the terminal adenosine overhang TA Cloning Vectors
- 17. The pGEM -T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme digestions for release of the insert. pGEM -T and pGEM -T Easy
- 18. The only difference between pGEM-T and pGEM-T Easy is in the multiple cloning site (MCS). The MCS of the pGEM-T Easy Vector contains sequences on either side of the insert that are recognized by the restriction enzymes Not I and EcoR I. This allows the insert DNA to be removed with a single restriction digest using either of these enzymes.
- 20. pCAMBIA
- 21. high copy number in E.coli for high DNA yields pVS1 replicon (replicon of the 29kbp of Pseudomonas plasmid pVS1) for high stability in Agrobacterium small size, 7-12kb depending on which plasmid restriction sites designed for modular plasmid modifications and small but adequate poly-linkers for introducing your DNA of interest bacterial selection with chloramphenicol or kanamycin plant selection with hygromycin B or kanamycin simple means to construct translational fusions to gusA reporter genes.
- 22. GUS reporter system The activity of a promoter (in terms of expression of a gene under that promoter) either in a quantitative way or through visualization The technique is based on β-glucuronidase, an enzyme from the bacterium Escherichia coli When incubated with some specific colorless or non- fluorescent substrates, can transform them into colored or fluorescent products
- 23. There are different possible glucuronides that can be used as substrates, depending on the type of detection needed (histochemical, spectrophotometrical, fluorimetrical). The most common substrate for GUS histochemical staining is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc): the product of the reaction is in this case a clear blue color. Other common substrates are p-nitrophenyl β-D-glucuronide for the spectrophotometrical assay and 4-methylumbelliferyl- beta-D-glucuronide (MUG) for the fluorimetrical assay