Can someone please help explain in a step by step process how to do this problem and what it means? I do not understand what it is asking for exactly in regards to positive, negative, and the resistance gene and so forth...especially about the media being selective. What does being selective mean?
1) Pretend you are a microbiologist and need to grow E. cold strains of different genotypes. For the following bacterial genotypes, indicate which amino acids, carbohydrates, and antibiotics, should be added to a bacterial growth media to provide both a media that will allow the bacteria to live and also a media that is the most selective media possible for that particular strain of bacteria. Indicate whether an ingredient is required for survival of a particular strain, whether it is added to be selective for a particular strain, or both.
his=histidine, trp=tryptophan, gal=galactose, amp=ampicillin, lac=lactose
Ingredients available for the media:
Amino acids: histidine, tryptophan
Carbohydrates: galactose, lactose, glucose
Antibiotics: ampicillin, tetracycline
Bacterial Genotypes:
1. his+, gal-, amp^r, tets^s, trp-, lac-
2. his-, gal-, amp^r, tet^r, trp-, lac-
3. his-, gal-, amp^r, tet^r, trp+, lac+
4. his+, gal-, amp^s, tet^s, trp-, lac-
To solve this problem, we need to identify the amino acids, carbohydrates, and antibiotics that should be added to the bacterial growth media for each genotype. We also need to determine whether an ingredient is required for the survival of a particular strain or if it is added to be selective for a particular strain.
Let's break it down step by step:
Step 1: Understanding the terminology and symbols used in the genotypes.
- his+: It means that the strain can synthesize histidine and does not require it in the media.
- his-: It means that the strain cannot synthesize histidine and requires it in the media for survival.
- gal-: It means that the strain cannot utilize galactose and does not require it in the media.
- amp^r: It means that the strain is resistant to ampicillin.
- tets^s: It means that the strain is sensitive to tetracycline.
- trp-: It means that the strain cannot synthesize tryptophan and requires it in the media.
- lac-: It means that the strain cannot utilize lactose and does not require it in the media.
- amp^s: It means that the strain is sensitive to ampicillin.
- tet^r: It means that the strain is resistant to tetracycline.
- trp+: It means that the strain can synthesize tryptophan and does not require it in the media.
- lac+: It means that the strain can utilize lactose and does not require it in the media.
Step 2: Determine the required and selective ingredients for each genotype.
For each genotype, we need to determine the appropriate ingredients to support the survival of the strain and select for that particular strain. Here's a breakdown:
Genotype 1: his+, gal-, amp^r, tets^s, trp-, lac-
- Required ingredients: histidine (because it is his-) and glucose (as a general carbon source)
- Selective ingredient: ampicillin (to select for the resistance gene)
- No lactose or galactose is required since the strain cannot utilize them.
Genotype 2: his-, gal-, amp^r, tet^r, trp-, lac-
- Required ingredients: histidine (because it is his-) and glucose (as a general carbon source)
- Selective ingredient: none needed, as both antibiotic resistance genes are present.
- No lactose or galactose is required since the strain cannot utilize them.
Genotype 3: his-, gal-, amp^r, tet^r, trp+, lac+
- Required ingredients: glucose (as a general carbon source)
- Selective ingredient: ampicillin (to select for the resistance gene)
- Lactose is required since the strain can utilize it.
- No histidine or galactose is required since the strain cannot synthesize histidine and cannot utilize galactose.
Genotype 4: his+, gal-, amp^s, tet^s, trp-, lac-
- Required ingredients: histidine (because it is his-)
- Selective ingredient: none needed, as both antibiotic-sensitive genes are present.
- No lactose or galactose is required since the strain cannot utilize them.
Step 3: Understanding selective media.
Selective media is designed to promote the growth of certain organisms while inhibiting the growth of others. In this context, media that is selective for a particular strain means that it contains ingredients (such as antibiotics) that only allow the growth of strains carrying specific genes (e.g., antibiotic resistance genes) while inhibiting the growth of strains without those genes. It helps in selectively isolating specific strains from a mixture of bacterial cultures.
In summary, to create the appropriate media for each genotype, you need to include the required ingredients (such as histidine, glucose, and lactose) for the survival of the strain and the selective ingredients (such as ampicillin) to select for the presence of specific genes. The selective media is designed in a way that allows the desired strains to grow while inhibiting the growth of undesired strains or contaminating organisms.