3. Next Generation Synthesis
Joseph Jacobson (MIT)
Class assignments
Synthetic oligonucleotides (oligos) enable us to assemble novel DNA sequences or create copies of existing genes. This week's assignment will give you experience building genes and expressing them in bacteria.
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Lab Task: Recode green fluorescent protein (GFP) to instead emit blue or cerulean light. Confirm success by transforming your assembled BFP and CFP variants into E coli cells.
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Theory Task: Design a gene. Describe a detailed workflow for constructing and expressing it. Identify how the parts of your genetic construct relate to DNA replication and the Central Dogma of Molecular Biology.
Next generation synthesis
27th of September - Joseph Jacobson (MIT)
We obteined the a plasmid sequence from https://www.addgene.org/vector-database/2623/ in GBK format.
The pET102 because we increased translation efficiency and solubility of heterologous proteins using N-terminal His-Patch thioredoxin.
We select the sities restriction to enter the prefer gene.
Beteween the SacI and HindIII we into gene sequence, but we check the frame with 6xHIS for purified protein in the future.
For present study we use the sequence of Synthetic construct Bevacizumab light chain gene, complete cds, we select this sequence because is part of bevacizumab a monoclonal antibody that blocks angiogenesis by inhibiting vascular endothelial growth factor A (VEGF-A) and its used to treatment to breast and lung cancer patients and have a cost of $100,000 a year in the United States.
Accession light chain:
KX119516.1
Accession
Accession: heavy chain
KX119517.1
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The oligos for used are T7 forward y reverse.
When we run PCR with T7 oligos, we will observe a fragment of size 1566pb.
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Next, we
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