Bacterial Mapping
Introduction (PARAGRAPH FORM)
-Genotypes and phenotype of bacteria
-introduce the organism, why use E.coli – describe the 2 strains: Hfr and F(-) and why we use them
CSH 121 (Hfr) : StrS, Leu+, His+, Arg+, Ade+, Trp+, TetR (donor)
CSH 125 (F-) : StrR, Leu-, His-, Arg-, Ade-, Trp-, TetS (recipient)
-Describe media used: complete, minimal, drop-out, antibiotic
-describe prototrophic and auxotrophic
-mechanism of bacterial transfer, focus on conjugation: describe F factor, origin of replication and terminus
-what is a colony
-what is serial dilution
Diluent
Stock solution
-what is bacterial “patching” and what it achieves
-what is percent linkage, include formula and what is map position
-what is the goal: to determine percent linkage and map position to determine which order genes are transferred during conjugation and if this matches published map
-Why is bacteria is great model organism for genetics; Discuss the types of bacteria – prototrophs, auxotrophs and what makes them special
– Possible media types and how we can use these types of media to identify strains (mutants and WT).
-Discuss the two strains used in the lab – recipient and donor (Hfr)– discuss their genotypes and how we used to genotype to do our selection.
-Discuss the three types of DNA transfer – focusing on conjugation.
Procedure
Week 1: Mating of CSH 121 and CSH 125 Bacteria
Three Eppendorf tubes were labelled with the group’s initials and: (1) CSH 121/CSH 125, (2) CSH 121, (3) CSH 125.
A p200 pipette and sterile pipette tip was used to transfer 200 uL of CSH 121 to each of the CSH121 and CSH 121/CSH 125 tubes.
A p200 pipette and sterile pipette tip was used to transfer 200 uL of CSH 125 to each of the CSH125 and CSH 121/CSH 125 tubes.
Lid was placed back on the bacterial stock tube and Eppendorf tubes as soon as possible after use to minimize contamination. Each pipette tip used in the transfer of liquids were disposed in the biohazard trash.
The tubes were placed in a 37 ℃ water bath for one hour.
Three Falcon tubes containing 2mL of LB were labelled with the group’s initials and: (1) CSH 121/CSH 125, (2) CSH 121, (3) CSH 125.
The Eppendorf tubes containing bacteria were retrieved after one hour of incubation. 200 uL from each of the Eppendorf tubes were transferred to the correct Falcon tube (identically labelled).
The three Falcon tubes were placed into a 37 ℃ shaking incubator for one hour. Steps 9-10 were done while the tubes were incubating.
The bottom side of six petri dishes containing streptomycin (+Strep) and no leucine were labelled accordingly: CSH 121/125 1:10, CSH 121/125 1:100, CSH 121/125 1:1000, or CSH 121/125 1:10,000, CSH 121 and CSH 125. The last two plates served as controls.
Eight Eppendorf tubes were labelled accordingly: 1/10, 1/100, 1/1,000 and 1/10,000 for the mating experiment and CSH 121 1/10, CSH 121 1/100, CSH 125 1/10 and CSH 125 1/100 for the controls.
Serial dilution was made according to the table below:
1/10
1/100
1/1000
1/10000
Experiment tube
10 uL
10 uL
10 uL
10 uL
Minimal media
90 uL
90 uL
90 uL
90 uL
Final volume
90 uL
90 uL
90 uL
100 uL
90 uL of bacteria from each dilution tube was transferred onto the appropriately labelled petri dish and spread over the surface of the agar media using a bent glass rod. Plates were allowed to dry for several minutes before stacking on top of each other (bottom agar side up) for incubation at 37 ℃ until next lab meeting.
Week 2: Replica Plating
Transformation plates from last week were estimated for amount of growth on each plate and recorded on the data page.
Six drop-out media plates 5 of which are missing one of the following amino acids: Leucine, Arginine, Histidine, Tryptophan or Adenine and one with Tetracycline were labelled around the perimeter with the group’s name. A template was placed over the bottom of the drop-out media to facilitate patching of colonies from the transformation plates onto the drop-outs.
Individual colonies were selected from plates with colonies that were not overlapping and plated onto the drop-out plates for a total of 30 colonies. Leu- drop-out media was plated last.
Plates were stacked on top of each other, inverted and placed on the instructor’s table for incubation.
Week 3: Data Analysis
Plates from the replica plating were retrieved and number of colonies that grew on each plate were recorded. Colonies that did not grow on the last leucine minus plate were excluded from the counts. Data was reported to the class.
Plates were returned to the instructor’s table for proper disposal.
The class’s pooled data was used to calculate the percent of linkage and then graphed in a semilog paper. “Best fit” line was drawn .
Graph from Step 3 was used to plot the remaining data points to determine the minutes for each gene.
A map of the gene order for the Hfr strain CSH121 was drawn.
Data Page
Week 2 Replica plating analysis: Growth on plates
Media Plate
Number of colonies
CSH121/CSH125 1/100
500+
CSH121/CSH125 1/1,000
74
CSH121/CSH125 1/10,000
11
CSH121
0
CSH125
0
Week 3 Data Analysis: Number of colonies present on each plate
Drop-out media plate
Number of colonies
His-
1
Arg-
2
Ade-
12
Trp-
3
Leu-
30
+Tet
18
Figure 1. Replication plates from Week 2
Figure 1. Replication plates from Week 2
Figure 2. Semilog paper showing map position (in min) vs percent linkage
Figure 2. Semilog paper showing map position (in min) vs percent linkage
Percent linkage
(His and Leu given at 4.8% and 100% , respectively)
Map position
Figure 3. Published map and class data showing genetic mapping from oriT
Figure 3. Published map and class data showing genetic mapping from oriT
21 mins 17 mins 15 mins 5 min 5 mins 2 mins
(69 mins) (44 mins) (28 mins) (12 mins) (5 mins) (2 mins)
Arg His Trp Ade Tet Leu
oriT
Published map
Class data
21 mins 17 mins 15 mins 5 min 5 mins 2 mins
(65 mins) (44 mins) (27 mins) (12 mins) (7 mins) (2 mins)
Arg His Trp Ade Tet Leu
oriT
21 mins 17 mins 15 mins 5 min 5 mins 2 mins
(69 mins) (44 mins) (28 mins) (12 mins) (5 mins) (2 mins)
Arg His Trp Ade Tet Leu
oriT
Published map
Class data
21 mins 17 mins 15 mins 5 min 5 mins 2 mins
(65 mins) (44 mins) (27 mins) (12 mins) (7 mins) (2 mins)
Arg His Trp Ade Tet Leu
oriT
Discussion (PARAGRAPH FORM)
Week 1
Why are we selecting for Leu+ and StrR colonies?
0 colonies on CSH 121 and 125 means controls are valid
What is genotype of Hfr strain CSH 121 and if it is prototroph or autotroph
What is genotype of F- strain CSH 125 and if it is prototroph or autotroph
Week 2
Why Leu- plate is last? Bc we are transferring from Leu+ plate, serves as control? Test valid? (Leu- plate should have NO GROWTH)
Interpret the result, is the result valid (NO GROWTH in controls)
Refer to Figure 1
Were we able to obtain 30 colonies—YES
What Leu- media select for? Leucine
Role of streptomycin? Inhibit growth of parent so only conjugates are selected
Why CSH121 doesn’t grow on these plates
Why CSH125 doesn’t grow on these plates
Week 3
Interpret figure 3
Percent linkage was determined using the formula and used to extrapolate the map position
Is semilog linear or logarithmic, why?
Did we achieve the goal of determining % linkage?
How this correlates to the published map?
What is linkage of the genes in SCH 121 (Hfr strain)?
Obtaining 30 colonies on Leu- plate means result is valid, all 30 colonies should be counted
Restate the purpose of the lab. week
What does the growth or no growth of the colonies indicate? Can any conclusions be made about any of the genes – where they all transferred? How can you determine this?
What is the answer to the question or objective(s) being explored – what is the order of the genes?
Discuss any problems you had with the lab: contamination, no colonies on dilution plates, no growth on drop-out plates.
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