Bacteria

Question 1

plasmid structure

Answer 1

-typically or always will have origin of replication where the plasmid will fire to let it be copied in bacterium and be maintained
-antibiotic resistance gene or other selectable marker- select for cells that have plasmid
-multiple cloning sites with gene of interest cloned into it
-in naturally occurring plasmids, there may be additional elements for very low copy number of plasmids like partitioning systems to help maintain them so they’re not lost after division, origin of transfer (conjugation), reporter genes, genes for replication

Question 2

origin of replication (ori)

Answer 2

determiens copy number and host range

Question 3

transformation

Answer 3

electroporation or heat shock to shock bacterial cells and get plasmid or other piece of DNA into them

Question 4

conjugation

Answer 4

using naturally occurring gene transfer to move plasmid from one cell to another

Question 5

transduction

Answer 5

leverage bacteriophage or virus to pick up pieces of DNA and let phage transfer it into recipient cell

Question 6

conjugation

Answer 6

-conjugation machinery in donor cell
-oriT/bom on plasmid
-susceptible recipient
-enzymes that will allow for nicking and transfer of the plasmid DNA from donor to the recipient

Question 7

engineering plasmid to ID genetically tractable gut bacteria

Answer 7

transform plasmid into E. coli donor –> mix donor with bacteria that have been extracted from human stool –> plate them either human alone, bacteria alone, or mixed population and plate them on medium that’ll allow us to successfully select or isolate bacteria who have taken up plasmid

Question 8

what should be included in the media to select for recipients that pick up plasmid?

Answer 8

include in the antibiotics that are specific to the selectable marker to select for transconjugates and select against E. coli with some selectable media that favors firmicutes

Question 9

allele exchange: suicide vector

Answer 9

-kan^R is the selectable marker
-sacB is the counter-selectable marker
-used in many bacteria to engineer the genome- make a specific change like a deletion, a mutation, or to integrate or replace particular locus with something we would want on there

Question 10

allele exchange

Answer 10

-allows us to exchange the WT gene in the chromosome with mutant or deletion at its native locus
-suicide vector has region of homology to the region of the genome we want to change and put into cells of interest then rely on host homologous recombination machinery to allow for plasmid to recombine into chromosome

Question 11

why does the plasmid for allele exchange have to integrate into the chromosome?

Answer 11

1. non-functional ori (but has a functional ori in E. coli that allows it to replicate)
2. selection for selectable marker

Question 12

first recombination event of allele exchange

Answer 12

use selectable marker that’s on the plasmid (kanamycin resistance) to select for those that have integrated this plasmid into the genome

Question 13

second recombination event for allele exchange

Answer 13

-we have two copies of gene of interest with the WT and mutated version as well as everything else in the plasmid (kanamycin and sacB)
-utilize the counter-selectable marker, or sacB, that causes the sucrose to be toxic –> grow the cells from the first recombination in sucrose without the antibiotic to select against the sacB gene and the cells that have lost the plasmid will survive
-need to exclude the kanamycin from the media for the second recombination since the mutant gene won’t have the kanR cassette to protect it

Question 14

how do you confirm that the plasmid has been successfully integrated and the change was made?

Answer 14

-depends on the size of the deletion
-large deletion –> DNA gel or western blot
-small deletion –> amplified with PCR and sequence to confirm

Question 15

comparative transposon sequencing (Tn-Seq)

Answer 15

-transposable element that can pop into genome
-in this method, introducing the transposon into a WT strain vs mutant strain and look to see if there is a difference in the essentiality of genes across the genome in these two conditions
-mutagenize –> select for mutants that carry a selectable marker with the transposon
-if it inserts somewhere that’s essential for growth, we would never get it –> any colonies that we get is a transposon insertion somewhere in genome that we can disrupt with and still allow for growth
-amplify from transposon to ID the insertion site and then map all of the insertion sites back to the genome across the entire genome

Question 16

looking for essentiality of a gene in WT vs mutant backgrounds

Answer 16

-conditionally essential- if the gene has transposons in WT but not in the mutant
-essential- no transposons in WT or mutant background
-non-essential- transposons in both WT and mutant backgrounds

Question 17

conditional gene overactivation

Answer 17

-inducible expression can be accomplished by placing the GOI behind an inducible promoter and this promoter can be turned on by presence of small molecule like IPTG, in a dose dependent manner
-affected by inducer concentrations
-gene under control of inducible promoter separate on plasmid that will be a replicating plasmid
-you can also have it on an integrating plasmid –> if you wanted to integrate plasmid into genome not at its native locus and have it be under control of inducible promoter
-you can also get rid of the gene @ its native locus and replace promoter with inducible promoter to drive overexpression of gene
-could also add a replicating plasmid that will add additional copies of the GOI

Question 18

degradation and overexpression with integrating plasmid

Answer 18

-both involve allele exchange with the plasmid of interest
-overactivation: place the GOI downstream of an inducible promoter that will do allele exchange to integrate the plasmid into the genome not @ its native locus then add IPTG in dose dependent manner
-degradation: tag native copy in the chromosome of your gene with ssrA tag and place ssrB adaptor under control of inducible promoter then grow up cells without inducer so that gene product will be produced then add inducer to turn on ssrA gene transcription and induce degradation of GOI

Question 19

another way to induce depletion of essential protein

Answer 19

inhibit transcription with CRISPRi that used dead cas9 to turn off transcription of the gene