Add Laboratory in Biotechniques II notes

13 - Spring 2024/Laboratory in Biotechniques II/001 - Project Introduction.md

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+> # 001 - Project Introduction
+> Class Notes - January 12, 2024
+> Emilio Soriano Chávez
+> ***
+> <span style="color:#117a65">Laboratory in Biotechniques II</span>
+> Spring 2024
+
+***
+
+- **Expression of Proteins in Bacteria:**
+	- Demetra (Demt)
+	- Focal Adhesion Kinase Catalytic Domain (FAKcd)
+	- Pectinase (PGA)
+
+- **Tasks:**
+	- Make gene expression vetor.
+	- Induced expression in bacteria.
+	- Purify expressed protein from bacteria.
+	- Characterize by SDS-PAGE / Western Blot analysis.
+
+***
+## Project 01 - Expression of Demt
+
+- Very large protein (~81.7 kDa).
+
+## Project 02 - Expression of FAKcd
+
+- Protein of 28.5 kDa.
+
+## Project 03 - Expression of PGA
+
+- Estimated molecular weight of ~36 kDa.
+
+***
+
+- Positive controls to choose from:
+	- RFP
+	- GFP
+
+***
+
+- pET28b Bacteria Expression Vector
+- Clone genes into expression vector.
+	- Between NcoI and XhoI restriction sites.
+- Deliver into *E. coli*.
+
+- His tag for detection.
+	- Anti-His to detect protein expression.
+- T7 promoter.
+
+- BL21 (DE3):
+	- DE3 construct in the genome of the bacteria.
+		- Its product is the T7 RNA Polymerase.
+	- Protease-deficient strand.
+
+- If we use other promoter, like `lac`(an endogenous promoter), we can use any bacteria strain.
+	- Expression may not be as high.
+
+***
+## Experimental Procedures
+
+- **Construction of Expression Vector:**
+	- Molecular Cloning
+- **Induced Protein Expression and Purification:**
+	- IPTG induces expression.
+	- Protein purification (His tag column).
+		- Wash column and elude column.
+- **Characterization of Recombinant Protein:**
+	- Electrophoresis
+	- Western Blot
+
+***
+
+- **Major Expression Systems:**
+	- Mammalian
+	- Yeast
+	- Bacteria (*E. coli*)
+		- Fast growth and fast expression.
+		- Cheap media compared to mammalian cells.
+		- Good knowledge of the host.
+		- Easy to genetically manipulate (plasmid delivery through heat shock method).
+		- **Challenges:**
+			- May not be able to produce larger proteins (>50 kDa).
+			- Cannot perform post-translational modification (like glycosylation).
+
+> [!INFO]
+> Insulin was the first protein recombinantly produced. Done by Genentech.
+
+## T7 Expression System
+
+- DE3 genes produce T7 RNA Polymerase.
+- **Vector:**
+	- T7 Promoter
+	- lac Operator (lacO)
+	- Target Gene
+	- Selection Marker (Kan)
+	- lacI
+		- Produces the repressor protein.
+		- lacO is bound by repressor in normal conditions so that transcription does not happen.
+
+- **pET28b:EGFP**
+	- T7 Promoter
+	- lacO
+	- RBS
+	- EGFP
+	- Terminator
+
+- Use NcoI and XhoI to cut and introduce our target genes.
+- PCR to amplify our target genes.
+- Design primers for PCR, which need to include the restriction sites.
+
+
+- lacI encodes repressor.
+	- Under normal conditions, the repressor binds lactose.
+	- Transcription will not happen until we add an inducer (lactose or IPTG).
+	- When lactose binds the repressor, the repressor will *fall off* from the lactose operon and transcription will happen.
+	- Because lactose is very expensive, we use IPTG (similar structure) that can bind to the repressor.
+
+- **IPTG induced expression in E. coli**
+
+> [!INFO]
+> When there is no glucose in bacterial media, they use the lactose operon to produce $\beta$ galactosidase to degrade lactose into galactose and glucose.
+
+- **Other T7 Expression Vectors:**
+	- pEX Vectors from OriGene
+	- pET/D-TOPO vector from Invitrogen
+
+- pGEX-4T-2
+	- Not necessary to have DE3 because there is no T7 promoter.
+
+> [!TODO]
+> - [ ] Design primers before Monday (Pectinase).