Question

Dr. Kenmore has continued his work and built new plasmids based off the pCK plasmids. There are some features common to all of them:

• ori: the origin of replication
• P_lacZ: The promoter for the lac operon. It is negatively regulated by LacI
• lacI: the negative regulator of P_lacZ
• bla: a gene that encodes ampicillin resistance

pCK:lacZ	pCK:gfp
pCK:Zcab	pCK:Gcab

The genes that vary on the plasmids are:

• lacZ: encodes beta-galactosidase, it cleaves lactose into glucose and galactose, or cleaves x-gal to make indole (blue color) and galactose.
• gfp: encodes green fluorescent protein this protein glows green under a UV light.
• pmoCAB: the operon that makes methane monooxygenase, the enzyme that converts methane and oxygen to methanol.

Dr. Kenmore has built the  pCK:Zcab and pCK:Gcab plasmids so that lacZ or gfp will be transcribed on the same mRNA as pmoCAB, making new synthetic operons the pmoCAB-lacZ operon and the pmoCAB-gfp operon

Dr. Kenmore wants to transform an E. coli strain that before transformation is ampicillin sensitive, and does not have lacZ on the chromosome. Dr. Kenmore wants to save a few steps and wants to know if any of the following media could be used for selection of these plasmids, and be simultaneously be used to show expression of genes from the plasmids. For each cell in the Table 1 and Table 2, pick if you think this combination of genotypes and media “selects for” the transformants (only the transformed cells cells grow) or “selects against” the transformants (the transformed cells will die on this media), or “not selective” if untransformed and transformed cells grow on this media.

Make sure you scroll left/right to make sure you are filling in every cell on all the tables.

Table Abbreviations

• MM – Minimal media: a mix of essential inorganic salts to support the growth of bacteria
• MAC – MacConkey Agar
• Amp – Ampicillin: a beta-lactam antibiotic

Table 1

	E. coli pCK:lacZ	E. coli pCK:gfp
MM + Lactose + X-gal	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]
MAC + Amp	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]
TSA + Lactose	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]
MM + Glucose + Amp + X-gal	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]

Table 2

	E. coli pCK:Zcab	E. coli pCK:Gcab
MM + Lactose + X-gal	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]
MAC + Amp	[ Select ]      [“Selects for Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]
TSA + Lactose	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]	[ Select ]      [“Selects for Transformants”, “Selects Against Transformants”, “Not Selective”]
MM + Glucose + Amp + X-gal	[ Select ]      [“Selects for Transformants”, “Selects Against Transformants”, “No Selection”]	[ Select ]      [“Selects For Transformants”, “Selects Against Transformants”, “Not Selective”]

In the table 3 and 4 below, describe the colony phenotypes you predict to see. You have a UV light at your disposal, you would wave it over the transformants, if they needed it or not. If you selected “Selects Against”, or “Not Selective” make the same selection here. (Dead cells have no colony phenotypes, and if untransformed and transformed cells grow, there’s a lawn of too many bacteria to count, so no colony phenotypes.)

Make sure you scroll left/right to make sure you are filling on all the tables.

Table 3

	E. coli pCK:lacZ	E. coli pCK:gfp
MM + Lactose + X-gal	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]
MAC + Amp	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]
TSA + Lactose	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]
MM + Glucose + Amp + X-gal	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Growth”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/Not Selective”]

Table 4

	E. coli pCK:Zcab	E. coli pCK:Gcab
MM + Lactose + X-gal	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]
MAC + Amp	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]
TSA + Lactose	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]
MM + Glucose + Amp + X-gal	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]	[ Select ]      [“Blue Colonies”, “White Colonies”, “Glowing Colonies”, “Pink Colonies”, “Selects Against/No Selection”]