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--- chapter_12 [2024/09/01 17:45] – [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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+<-chapter_11|Chapter 11^table_of_contents|Table of Contents^chapter_13|Chapter 13->
 <typo fs:x-large>%%Chapter 12. Cloning regulated genes in eukaryotes%%</typo>
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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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 </figure>
-Now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into //S. cerevisiae// cells that are mutant for the $ura3$ gene (these cells are uracil auxotrophs). The method for transformation and preparing competent yeast cells is similar to that of preparing competent //E. coli//, except that LiCl is used instead of CaCl<sub>2</sub> to treat the cells. Importantly, the fragments cannot replicate on their own, but they can integrate into the chromosome through homologous recombination that targets DNA sequences on the chromosome that that match the transformed fragments. After transformation, we grow the cells on plates that lack uracil - we are selecting for cells that have taken up a $URA3$-containing DNA fragment somewhere in its genome. Each Ura<sup>+</sup> transformant colony that grows will have recombined a mini-Tn7-containing genomic DNA fragment into its genome. This essentially gives us a library of yeast with transposons randomly integrated into its genome.
+Now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into //S. cerevisiae// cells that are mutant for the $ura3$ gene (these cells are uracil auxotrophs). The method for transformation and preparing competent yeast cells is similar to that of preparing competent //E. coli//, except that LiCl is used instead of CaCl<sub>2</sub> to treat the cells. Importantly, the fragments cannot replicate on their own, but they can integrate into the chromosome through homologous recombination that targets DNA sequences on the chromosome that match the transformed fragments. After transformation, we grow the cells on plates that lack uracil - we are selecting for cells that have taken up a $URA3$-containing DNA fragment somewhere in its genome. Each Ura<sup>+</sup> transformant colony that grows will have recombined a mini-Tn7-containing genomic DNA fragment into its genome. This essentially gives us a library of yeast with transposons randomly integrated into its genome.
 <figure Fig6>
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 </figure>
-Note that the $lacZ$ gene in the transposon contains only its amino acid coding sequence. It does not have its own regulatory sequences from //E. coli// such as $lacO$ and $lacP$ (and even if $lacO$ and $lacP$ were present, it would have no effect on $lacZ$ expression in yeast; think about that!). But if the transposon inserts downstream of a yeast gene promoter (somewhat rare) and in the correct orientation (1 in 2 chance), and in the correct triplet codon reading frame (1 in 3 chance), the lacZ gene would then come under the control of that promoter. When transcription is activated from that promoter, a LacZ fusion protein is expressed, and most LacZ fusion proteins have robust β-galactosidase activity.
+Note that the $lacZ$ gene in the transposon contains only its amino acid coding sequence. It does not have its own regulatory sequences from //E. coli// such as $lacO$ and $lacP$ (and even if $lacO$ and $lacP$ were present, it would have no effect on $lacZ$ expression in yeast; think about that!). But if the transposon inserts downstream of a yeast gene promoter (somewhat rare) and in the correct orientation (1 in 2 chance), and in the correct triplet codon reading frame (1 in 3 chance), the $lacZ$ gene would then come under the control of that promoter. When transcription is activated from that promoter, a LacZ fusion protein is expressed, and most LacZ fusion proteins have robust β-galactosidase activity.
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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:
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   - to identify genes whose transcription is upregulated in response to being exposed to this tobacco smoke chemical.
-The chemical we will use as an example is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Yeast cells with random mini-Tn7 insertions are first plated out on Petri dishes at a low density so that individual cells each give rise to single colonies on the agar surface. Each colony represents a clone that contains a unique insert. To screen the library for genes that protect against NNK-induced cell killing, the colonies are replica-plated onto agar medium that either does or does not contain a high dose of NNK. Replica plating is a technique where colonies on one Petri dish are copied to a different Petri dish while maintaining their positions in the dish, so that we can the observe the same clone under different growth conditions. To screen the library for genes that are transcriptionally regulated in the presence of NNK, the colonies are replica-plated onto agar medium containing either X-gal alone or X-gal plus a low dose of NNK.
+The chemical we will use as an example is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Yeast cells with random mini-Tn7 insertions are first plated out on Petri dishes at a low density so that individual cells each give rise to single colonies on the agar surface. Each colony represents a clone that contains a unique insert. To screen the library for genes that protect against NNK-induced cell killing, the colonies are replica plated onto agar medium that either does or does not contain a high dose of NNK. Replica plating is a technique where colonies on one Petri dish are copied to a different Petri dish while maintaining their positions in the dish, so that we can the observe the same clone under different growth conditions. To screen the library for genes that are transcriptionally regulated in the presence of NNK, the colonies are replica plated onto agar medium containing either X-gal alone or X-gal plus a low dose of NNK.
 <figure Fig7>