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--- chapter_12 [2025/04/10 12:38] – [Creating an insertion library for yeast] mike
+++ chapter_12 [2025/04/15 06:57] (current) – [Creating an insertion library for yeast] mike
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   * A library of yeast genomic fragments cloned into a bacterial plasmid. We learned about the concept of genomic libraries in [[chapter_09|Chap. 09]].
   * The //E. coli// $lacZ$ gene. We learned about the $lacZ$ gene in Chapters [[chapter_09|09]] and [[chapter_10|10]]. In this experiment the $lacZ$ gene is going to be used in yeast cells as a reporter gene (sometimes just called a reporter) for transcriptional activity of yeast genes. The $lacZ$ coding sequence works in yeast because //E. coli// and yeast both use the exact same universal genetic code for converting triplet codon sequences into amino acids.
-  * A modified bacterial transposon called mini-Tn7 (Fig. {{ref>Fig3}}). Transposons are naturally occurring pieces of DNA that can transpose, or jump around, to random locations in genomes. We can modify transposons such that we can experimentally control when they jump around, and we can also construct them to carry genetic markers that help us track the transposon. In this experiment, we have engineered mini-Tn7 to contain the $lacZ$ gene (but without any cis-acting regulatory sequences such as $lacO$ or $lacP$), a yeast gene called $URA3$ required for uracil prototrophy (in this case we are including native upstream regulatory sequences necessary for yeast cells to express $URA3$, and an //E. coli// gene (plus appropriate bacterial regulatory sequences) that confers drug resistance to the antibiotic tetracycline ($tet^R$).
+  * A modified bacterial transposon called mini-Tn7 (Fig. {{ref>Fig3}}). Transposons are naturally occurring pieces of DNA that can transpose, or jump around, to random locations in genomes. We can modify transposons such that we can experimentally control when they jump around, and we can also construct them to carry genetic markers that help us track the transposon. In this experiment, we have engineered mini-Tn7 to contain the $lacZ$ gene (but without any cis-acting regulatory sequences such as $lacO$ or $lacP$), a yeast gene called $URA3$ required for uracil prototrophy (in this case we are including native upstream regulatory sequences necessary for yeast cells to express $URA3$), and an //E. coli// gene (plus appropriate bacterial regulatory sequences) that confers drug resistance to the antibiotic tetracycline ($tet^R$).
 <figure Fig3>
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   - Any transposon that integrated into a gene will essentially disrupt that gene and is likely to generate a null mutation (complete loss of function). Null mutants are very useful!
   - For transposons that integrate such that the $lacZ$ gene is in frame with the coding region of the yeast gene, the level of β-galactosidase (LacZ) activity in these cells therefore becomes an indicator) for the level of transcription of that gene. We call $lacZ$ in this context a reporter gene (sometimes we abbreviate that to just "reporter").
-  - This kind of insertion library approach allows you to use tricks like reverse PCR (see Fig. {{ref>Fig8}}) to help make cloning your gene easier (this method is much easier than cloning by complementation).
+  - This kind of insertion library approach allows you to use tricks like inverse PCR (see Fig. {{ref>Fig8}}) to help make cloning your gene easier (this method is much easier than cloning by complementation).
 Here are two examples of how such a library can be used: