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--- chapter_09 [2024/09/01 14:57] – [Cloning by complementation] mike
+++ chapter_09 [2025/03/18 18:04] (current) – [Gene Complementation in Bacteria: F plasmids] mike
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-<typo fs:x-large>Chapter 09. %%Complementation%% in bacteria</typo>
+<-chapter_08|Chapter 08^table_of_contents|Table of Contents^chapter_10|Chapter 10->
+<typo fs:x-large>Chapter 09. %%Complementation in bacteria%%</typo>
 In this chapter, we initially touch on some concepts in classical //E. coli// genetics that may not be of practical interest to all students except future microbiologists, such as F plasmids and Hfr mapping. However, it is useful to learn these concepts because later in the chapter we talk about cloning by complementation, which is a critical concept and skill that all serious students of biology (especially those interested in the area of molecular and cellular biology) need to understand.
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 {{ :f_plasmid.png?400 |}}
 <caption>
-F plasmids. //oriT// (erroneously labeled as OriT in the figure )is the origin of replication for the F plasmid, and the //tra// genes (erroneously labeled as Tra in the figure) are required for forming the pilus needed for transfer of the F plasmid from one cell to another (see Fig. {{ref>Fig2}}). F plasmids are about 10<sup>5</sup> bp long; for comparison, the size of the circular //E. coli// chromosome is 5x10<sup>6</sup> bp, or over 10-fold longer.
+F plasmids. //oriT// (erroneously labeled as OriT in the figure) is the origin of transfer (related to how F is transmitted from one host to another) for the F plasmid, and the //tra// genes (erroneously labeled as Tra in the figure) are required for forming the pilus needed for transfer of the F plasmid from one cell to another (see Fig. {{ref>Fig2}}). F plasmids are about 10<sup>5</sup> bp long; for comparison, the size of the circular //E. coli// chromosome is 5x10<sup>6</sup> bp, or over 10-fold longer.
 </caption>
 </figure>
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 ===== F' is a version of F that carries segments of the $E. coli$ chromosome =====
-Homologous recombination can sometimes occur at a different position to give excise an F plasmid that carries a part of the //E. coli// chromosome. In the example in Fig. {{ref>Fig6}}, this is the BC segment of the //E. coli// chromosome. This form of F is called an F' (pronounced “F prime”).
+Homologous recombination can sometimes occur at a different position to excise an F plasmid that carries a part of the //E. coli// chromosome. In the example in Fig. {{ref>Fig6}}, this is the BC segment of the //E. coli// chromosome. This form of F is called an F' (pronounced “F prime”).
 <figure Fig6>
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 {{ :r_plasmid.png?400 |}}
 <caption>
-An R factor that confers multiple drug resistance to bacteria.
+An R factor that confers multiple drug resistance to bacteria. Note that $sul^r$, $amp^r$, $kan^r$, and $tet^r$ are erroneously labeled as Sul<sup>r</sup>, Amp<sup>r</sup>, Kan<sup>r</sup>, and Tet<sup>r</sup>, respectively.
 </caption>
 </figure>
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 {{ :minimal_r_plasmid.png?400 |}}
 <caption>
-Schematic of a modified R plasmid used for cloning. For scale, a typical F plasmid is around 10<sup>5</sup> bp of DNA, but a typical R plasmid used for cloning is just a few thousand bp.
+Schematic of a modified R plasmid used for cloning. For scale, a typical F plasmid is around 10<sup>5</sup> bp of DNA, but a typical R plasmid used for cloning is just a few thousand bp. Note that $amp^r$ is erroneously labeled as Amp<sup>r</sup>.
 </caption>
 </figure>
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 {{ :cloning_by_complementation_e_coli.png?400 |}}
 <caption>
-Cloning by complementation from a DNA library generated from the //E. coli// genome.
+Cloning by complementation from a DNA library generated from the //E. coli// genome. Note that the $amp^r$ gene is erroneously labeled as Amp<sup>r</sup>.
 </caption>
 </figure>
-Say we wanted to clone the Lac operon, or genes from the Lac operon (more details on the Lac operon are discussed in [[chapter_10|Chap. 10]]). First, a genomic library would be made from chromosomal DNA from a Lac<sup>+</sup> //E. coli// strain using an R plasmid as our cloning vector (Fig. {{ref>Fig11}}). To make this library, //E. coli// chromosomal DNA (we can also say genomic DNA) is purified and cleaved with a restriction enzyme to fragment the chromosome. An appropriate R plasmid is cleaved with the same restriction enzyme, and the chromosomal fragments are ligated to the R plasmid. This library would then be used to transform a Lac<sup>–</sup> //E. coli// strain. Transformants (i.e., //E. coli// cells carrying a plasmid from the library) would first be selected for by resistance to the antibiotic ampicillin, the phenotype  (Amp<sup>R</sup>) of which is conferred by a gene on the R plasmid we are using ($ampR$). Any cell that is ampicillin resistant must carry a plasmid from the library. (As a side note, R plasmids have a property that make it so that each clone can only carry one type of R plasmid; therefore, each clone will contain a different and unique chromosomal fragment.) The resistant colonies would then be selected for the ability to grow on lactose (Lac<sup>+</sup>). These clones should not only contain R plasmids, but they should also carry an insert that contains a functional Lac operon.
+Say we wanted to clone the Lac operon, or genes from the Lac operon (more details on the Lac operon are discussed in [[chapter_10|Chap. 10]]). First, a genomic library would be made from chromosomal DNA from a Lac<sup>+</sup> //E. coli// strain using an R plasmid as our cloning vector (Fig. {{ref>Fig11}}). To make this library, //E. coli// chromosomal DNA (we can also say genomic DNA) is purified and cleaved with a restriction enzyme to fragment the chromosome. An appropriate R plasmid is cleaved with the same restriction enzyme, and the chromosomal fragments are ligated to the R plasmid. This library would then be used to transform a Lac<sup>–</sup> //E. coli// strain. Transformants (i.e., //E. coli// cells carrying a plasmid from the library) would first be selected for by resistance to the antibiotic ampicillin, the phenotype  (Amp<sup>R</sup>) of which is conferred by a gene on the R plasmid we are using ($amp^r$). Any cell that is ampicillin resistant must carry a plasmid from the library. (As a side note, R plasmids have a property that make it so that each clone can only carry one type of R plasmid; therefore, each clone will contain a different and unique chromosomal fragment.) The resistant colonies would then be selected for the ability to grow on lactose (Lac<sup>+</sup>). These clones should not only contain R plasmids, but they should also carry an insert that contains a functional Lac operon.
-How many clones would we need to screen? Depending on the type of restriction enzyme used, each plasmid carries about 4 x 10<sup>3</sup> bp of chromosomal DNA (the frequency of recognition sites is {(\frac{1}{4})}^6 for an enzyme like //EcoR//I that recognizes a 6 bp site, or one site every 4096 bp on average). The //E. coli// chromosome is 5 x 10<sup>6</sup> bp; thus, the entire //E. coli// genome will be covered if several thousand clones from the library are screened.
+How many clones would we need to screen? Depending on the type of restriction enzyme used, each plasmid carries about 4 x 10<sup>3</sup> bp of chromosomal DNA (the frequency of recognition sites is $(\frac{1}{4})^6$ for an enzyme like //EcoR//I that recognizes a 6 bp site, or one site every 4096 bp on average). The //E. coli// chromosome is 5 x 10<sup>6</sup> bp; thus, the entire //E. coli// genome will be covered if several thousand clones from the library are screened.
 All sorts of genes from //E. coli// have been cloned by looking for DNA fragments that can restore function to a mutant. It is also possible to find genes from other bacteria. The following is a dramatic example of a cloning experiment to find an important protein for a pathogenic bacterium.
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 ===== Questions and exercises =====
-Exercise 1: In Chap. {{ref>5}} we discussed the relationship between recombination frequency (m.u. or cM) and physical distance (measured in bp of DNA) (see also [[chapter_12|Chap. 12]] Fig. 1). What is the relationship between minutes in Hfr mapping and physical distance? In other words, how many bp of DNA is equivalent to 1 minute? See [[chapter_08|Chap. 08]] for some key information you might need.
+Exercise 1: In [[chapter_05|Chap. 05]] we discussed the relationship between recombination frequency (m.u. or cM) and physical distance (measured in bp of DNA) (see also [[chapter_12|Chap. 12]] Fig. 1). What is the relationship between minutes in Hfr mapping and physical distance? In other words, how many bp of DNA is equivalent to 1 minute? See [[chapter_08|Chap. 08]] for some key information you might need.
-Conceptual question: why would a strain with a mutated modifying enzyme but a wildtype restriction enzyme (R+ M-) be inviable (incompatible with life)? In other words, why is $R^+ / / M^-$ a lethal mutation?
+Conceptual question: why would a strain with a mutated modifying enzyme but a wildtype restriction enzyme ($R^+ M^-$) be inviable (incompatible with life)? In other words, why is $R^+ M^-$ a lethal combination of alleles?