分子生物学基本概念

[1]The Shine-Dalgarno sequence(AGGAGG), proposed by Australianscientists John Shine and Lynn Dalgarno,[1] is a ribosomal binding site located upstream of the start codon AUG. It is a consensus sequence that helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon. The complementary sequence (CCUCCU), is called the anti-Shine-Dalgarno sequence and is located at the 3' end of the 16S rRNA in the ribosome.Mutations in the Shine-Dalgarno sequence can reduce translation. This reduction is due to a reduced mRNA-ribosome pairing efficiency, as evidenced by the fact that complementary mutations in the anti-Shine-Dalgarno sequence can restore translation.When the Shine-Dalgarno sequence and the anti-Shine-Dalgarno sequence pair, the translation initiation factors IF2-GTP, IF1, IF3, as well as the initiator tRNA fMet-tRNA(fMET) are recruited to the ribosome.Shine-Dalgarno sequence vs. ribosomal S1 protein in Gram-negative bacteria, however, Shine-Dalgarno sequence presence is not obligatory for ribosome to locate initiator codon, since deletion of anti-Shine-Dalgarno sequence from 16S rRNA doesn't lead to translation initiation at non-authentic sites.Moreover, numerous prokaryotic mRNAs don't possess Shine-Dalgarno sequences at all. What principally attracts ribosome to mRNA initiation region is apparently ribosomal protein S1, which binds to AU-rich sequences found in many prokaryotic mRNAs 15-30 nucleotides upstream of start-codon. It should be noted, that S1 is only present in Gram-negative bacteria, being absent from Gram-positive species.SD序列（16S互补区）是位于原核生物mRNA 起始密码子（AUG）上游5~10个核苷酸处，一段富含嘌呤的序列。

其与核糖体小亚基中的16S rRNA的3’末端互补配对，促进mRNA 的翻译。

[2]ORF：An open reading frame (ORF) is a portion of a gene’s sequencethat contains a sequence of bases, uninterrupted by stop sequences, that could potentially encode a protein. When a new gene is identified and its DNA sequence deciphered, it is still unclear what its corresponding protein sequence is. This is because, in the absence ofany other knowledge, the DNA sequence can be translated or read in six possible reading frames (three for each strand，corresponding to three different start positions for the first codon). ORF identification involves scanning each of the six reading frames and determining which one(s) contains a stretch of DNA sequence bounded by a start and stop codon, yet containing no start or stop codons within it; a sequence meeting these conditions could correspond to the actual single product of the gene. The identification of an ORF provides the first evidence that a new sequence of DNA is part or all of a gene encoding for a particular protein.开放性阅读框架是结构基因上一段从起始密码子至终止密码子的核苷酸序列，其编码一个多肽或蛋白质。

通过ORF的检测识别可以判断一条新克隆的DNA序列是否是一个完整的基因。

[3]The blue-white screen is a screening technique that allows for thedetection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell (bacteria). The competent cells are grown in the presence of X-gal. If the ligation was successful, the bacterial colony will be white; if not, the colony will be blue. This technique allows for the quick and easy detection of successful ligation. β-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state. However, a mutant β-galactosidase derived from the M15 strain of E. coli has its N-terminal residues 11—41 deleted and this mutant, the ω-peptide, is unable to form a tetramer and is inactive. This mutant form of protein however may return fully to its active tetrameric state in the presence of an N-terminal fragment of the protein, the α-peptide.The rescue of function of the mutant β-galactosidase by the α-peptide is called α-complementation.In this method of screening, the host E. coli strain carries the lacZ deletion mutant (lacZΔM15} which contains the ω-peptide, while the plasmids used carry the lacZα sequence which encodes the first 59 residues of β-galactosidase, the α-peptide. Neither are functional by themselves.However, when the two peptides are expressed together, as when aplasmid containing the lacZα sequence is transformed int o a lacZΔM15 cells, they form a functional β-galactosidase enzyme.The blue/white screening method works by disrupting this α-complementation process. The plasmid carries within the lacZα sequence an internal multiple cloning site (MCS). This MCS within the lacZα sequence can be cut by restriction enzymes so that the foreign DNA may be inserted within the lacZα gene, thereby disrupting the gene and thus production of α-peptide. Consequently, in cells containing the plasmid with an insert, no functional β-galactosidase may be formed.The presence of an active β-galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional β-galactosidase. Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefor e no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.蓝白斑筛选是一种基因工程常用的重组菌筛选方法。