02 - Code Breakers: Using CRISPR to Rewrite Genetics
Experiment Summary CELLS:
BactoBeads E. coli pChromoPurple: The host bacteria have been genetically engineered to produce the purple protein. First, the E.coli genome has been modified to contain the gene for T7 polymerase under the control of the lac operon. Next, the modified bacteria are transformed with a plasmid containing the Purple Chromoprotein under the control of the T7 promoter. When IPTG is added, derepressing the lac operon, the cell can synthesize T7 polymerase. The poly- merase binds to the plasmid and turns on production of the purple protein. BactoBeads E. coli HB101: The host bacteria’s genome has been genetically engineered to express the beta-galacto- sidase protein under the control of the lac operon. When IPTG is added, derepressing the lac operon, the cell produces the beta-gal protein. This enzyme cleaves X-gal into bioprod- ucts that cause the colonies to change from white to blue. PLASMIDS: pCas-purple This plasmid contains the CAS gene, the chloramphenicol resistance gene, and a sgRNA that targets the purple Chromoprotein mRNA. pCas-Blue This plasmid contains the CAS gene, the chloramphenicol resistance gene, and a sgRNA that targets the beta-gal mRNA.
pCas-sgRNA
WHAT WILL HAPPEN? Write your hypotheses below. Set 1: E coli Chomopurple + pCas-purple
Target gene DNA
pCas-sgRNA
Set 2: E coli Chomopurple + pCas-blue:
Set 3: E coli HB101 + pCas-blue:
Set 4: E coli HB101 + pCas-purple:
Bacterial Colonies
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