[1] The Shine-Dalgarno sequence (AGGAGG), proposed by Australian scientists 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 poe 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 sequence that 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 of
any other knowledge, the DNA sequence can be translated or read in six poible 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 the detection of succeful 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 succeful, the bacterial colony will be white; if not, the colony will be blue.This technique allows for the quick and easy detection of succeful 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 expreed together, as when a
plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional β-galactosidase enzyme.The blue/white screening method works by disrupting this α-complementation proce.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 colourle 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 (therefore 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。野生型大肠杆菌产生的β-半乳糖苷酶可以将无色化合物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)切割成半乳糖和深蓝色的物质5-溴-4-靛蓝。有色物质可以使整个培养菌落产生颜色变化，而颜色变化是鉴定和筛选的最直观有效的方法。设计适用于蓝白斑筛选的基因工程菌为β-半乳糖苷酶缺陷型菌株。这种宿主菌的染色体基因组中编码β-半乳糖苷酶的基因突变，造成其编码的β-半乳糖苷酶失去正常N段一个146个氨基酸的短肽（即α肽链），从而不具有生物活性，即无法作用于X-gal产生蓝色物质。用于蓝白斑筛选的载体具有一段称为lacz\'的基因，lacz\'中包括：一段β-半乳糖苷酶的启动子；编码α肽链的区段；一个多克隆位点(MCS)。MCS位于编码α肽链的区段中，是外源DNA的选择性插入位点。虽然上述缺陷株基因组无法单独编码有活性的β-半乳糖苷酶，但当菌体中含有带lacz\'的质粒后，质粒lacz\'基因编码的α肽链和菌株基因组表达的N端缺陷的β-半乳糖苷酶突变体互补，具有与完
整β-半乳糖苷酶相同的作用X-gal生成蓝色物质的能力，这种现象即α-互补。操作中，添加IPTG(异丙基硫代-β-D-半乳糖苷)以激活lacz\'中的β-半乳糖苷酶的启动子，在含有X-gal的固体平板培养基中菌落呈现蓝色。以上是携带空载体的菌株产生的表型。当外源DNA（即目的片段）与含lacz\'的载体连接时，会插入进MCS（即靶基因），使α肽链读码框破坏，这种重组质粒不再表达α肽链，将它导入宿主缺陷菌株则无α互补作用，不产生活性β-半乳糖苷酶，即不可分解培养基中的X-gal产生蓝色，培养表型即呈现白色菌落。实验中，通常蓝白筛选是与抗性筛选一同使用的。含X-gal的平板培养基中同时含有一种或多种载体所携带抗性相对应的抗生素，这样，一次筛选可以判断出：未转化的菌不具有抗性，不生长；转化了空载体，即未重组质粒的菌，长成蓝色菌落；转化了重组质粒的菌，即目的重组菌，长成白色菌落。
[4]
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