Genetic Engineering (GE1) Lecture 7 PDF

Genetic Engineering (GE-1) Methodology of gene manipulation Dr. Mohamed Hazman Lecture 7 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 1  Promoters: screening and gene expression APBI303 (...

Genetic Engineering (GE-1) Methodology of gene manipulation Dr. Mohamed Hazman Lecture 7 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 1  Promoters: screening and gene expression APBI303 (Lecture 7) Dr. Mohamed Hazman 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 2  Reading vectors: basic elements Origin of replication (Ori) Multiple Cloning Site (MCS) Selectable Marker Promoter Sequence elements of translation 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 3  Vectors Cloning Expression Special elements for Making copies of gene Expressing protein protein expression and purification 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 4  Blue/white colony for cloning, not expression Clear and sharp color Empty vector separation reflect optimum IPTG and X-Gal applied amounts Recombinant vector Medium has been supplied with Amp + X-Gal + IPTG Courtesy: Mohamed Hazman, School of biotechnology, NU Academic year 2023-2024 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 5 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 6  to express a gene, you need a special E.coli (ex: Rosetta) and special vector (called expression vector) See next Chloramphenicol slide resistance gene Produce toxic protein, why? cells that harbor pHMGWA without the insert, will produce toxic protein and die. Unlike cells that harbor recombinant vector will survive and produce colonies, because no toxin protein will be produced 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 7  IPTG induces protein expression! (it is different story from blue/white colonies) Insert will be recombined between attR1 and attR2 (any genes between these two borders will be removed) IPTG dissociates repressor (a protein) from lac operator and “clears” the way to T7 RNA poly (expressed by GE bacteria, ex: BL21 and Rosetta) to bind promoter then T7 RNA pol enzyme will be expressed Image courtesy: M. Hazman transcribe GOI then from E.coli CS in case IPTG is added, expression then it binds to T7 promoter (genetically engineered E.coli) 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 8  Recombinant Protein tags (MBP) Maltose binding protein (MBP) is a common protein expression tag, as it is known to significantly enhance the solubility of many proteins. MBP is one of the most well-known and accomplished means of tagging proteins expressed in microbes. Fusion of a target protein to MBP permits its one-step purification using amylose resin. The MBP- fusion protein will bind to amylose resin while other proteins flow through. The MBP-protein fusion can then be eluted from the resin with maltose. The purified fusion protein can then be treated with a specific protease to cleave the MBP-tag from the target protein and the target protein can be separated from the MBP tag by affinity chromatography. 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 9  Recombinant Protein tags (6xHis tag) The histidine tag The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly-His-tag fused to its N- or C-terminus. Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions. In addition, anti-His-tag antibodies are commercially available for use in assay methods involving His-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein-specific antibody or probe. 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 10  Purification strategy Purified protein Before After using MBP trap Hazman et al (2019) 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 11  Inducible Promoters Ex2: Mammalian inducible gene expression system Ex: Tetracycline-controlled operator system (Tn10 tetracycline resistance operon) Tn is for transposon Three configurations: ֎Repression-based configuration Check also IPTG induction of ֎Tet-off configuration gene expression ֎Tet-on configuration 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 12  Tetracycline Tetracycline eliminate sensitive cells by binding to ribosome and thus mis-reads mRNA for protein synthesis E.coli strains as XL1-Blue or OmniMAX 2 T1R are tetracycline resistant by Tn10 transposon (TetR/TetA operons) Drug efflux 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 13  Repression-based configuration TetO (tetracycline operon) is inserted between the constitutive promoter and GOI TetO where the repressor (TetR) binds and thus inhibits gene expression At well, the addition of Dox (Doxycycline, analog for tetracycline) dissociates (disrupts) TetR-tetO, thus releasing RNA polymerase and initiating transcription 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 14  Tet-off configuration TRE: Tetracycline Response Element (7 repeats of 19 N of tetO) tTA: tetracycline-controlled transactivator The expression is ON in the absence of tetracycline At will, to pause the expression, added tetracycline will dissociate the chimeric protein rTA 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 15  Tet-on configuration Exogenous gene is NOT expressed as long as tetracycline is absent. Mutated tTA called rtTA (reversed tetracycline controlled transactivator) binds to TRE in the presence of tetracycline, thus, recruiting RNA polymerase, thus gene expression is on 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 16  Tissue-specific promoter: initiation Localized activity: tissue specific and cell-type specific Specific expression in a certain tissue or organ is normally due to the combined activities of cis- and trans-acting elements. Cis-regulatory elements (recognized) and Trans-acting proteins (TFs; repressors or activators of gene expression). Identifying the cis-acting elements responsible for tissue-specific gene expression is essential in gene therapy approaches The key is the Enhancer Elements: activation in specific tissues, even cell-type specific Enhancers cooperate with promoters to fine- Various transcription factors interact with cis acting tune gene expression (where/when/how elements in promoter regions and form a transcriptional much) initiation complex on the TATA box (core promoter) close to transcription initiation sites 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 17  Focused vs dispersed transcription binds Promoter contains elements responsible for its specific TF tissue-specific expression of ANY GENE Tissue specific Initiator: binds Housekeeping general TF 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 18  CpG and TATA box CpG islands TATA box Regions devoid of methylation 5′-TATAAA-3 ′ that have a higher G+C content Basic expression in tissue- than the genome average specific promoters. Less Generic transcriptional initiation accessible to chromatin. sites (no TATA is present) 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 19 Ex1: Tissue-Specific Promoters in Colorectal Cancer CEA: The third most prevalent type of Carcinoma cancer in the world Embryonic Antigen Gene suicide can potentiate classical cytotoxic effects Caspases: The main challenge is to find a proteas suitable vehicle to deliver the enzymes therapeutic gene safely and efficiently Tumor-specific promoters: Increase localized-base targeting Overexpressing therapeutic genes in tumor cells, not in normal cells 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 20  Potentiate specificity and targeting 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 21  Ex3: Endosperm specific glutelin promoter Directing the transgene expression in the endosperm of rice seeds. This would not be achieved by using regular constitutive promoters as they will cost or inherent negative embryo consequences such as retarded growth or stunted leaves. On the other hand, Tissue- Pro-vitamin A specific promoters can focus gene expression in the target edible part of the plant (seed) without unnecessary metabolic penalties. Regular endosperm 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 22 Thanks and good luck 13 November 2024 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 7 23