This was a study on identifying different mutant E. coli donor and recipient cells and determining the efficiency of conjugation between them. Different dilutions of donor and recipient cells were first plated on L-agar without chloramphenicol, L-agar with chloramphenicol, and MacConkey plates in order to identify them. The donor and recipient cells were then combined and allowed to conjugate. These transconjugate cells were plated on MacConkey plates in order to identify them and determine conjugation efficiency. Lastly, plasmids were isolated from the donor, recipient, and transconjugate cells. The DNA bands were mapped using electrophoresis. It was found that the donor cells could grow on the L-agar without chloramphenicol and on the MacConkey plates. On the MacConkey plates, they produced purple colonies. They could not grow on the L-agar with chloramphenicol. The recipient cells could grow on both L-agar plates, and on the MacConkey plates they yielded white colonies. The transconjugate cells produced both purple and white colonies on the MacConkey plate. The efficiency of conjugation was found to be 8.77%. The photographs of the DNA bands showed that the transconjugate cells had plasmids the same size as the donor cells, and that the recipient cells did not have a plasmid. In conclusion, the recipient and donor cells were both identified, and conjugation successfully produced transconjugates, as shown by the MacConkey plates and DNA bands.
There are three ways for bacteria to pass on genetic material: transformation, transduction, and conjugation (Russell, 2003). Conjugation is the main way for the bacteria E. coli to transfer its genetic material (Black, 1999). When transferring genetic material during conjugation, there must be a donor cell and a recipient cell (Hartwell, 2000). The donor cell contains an F plasmid and the recipient cell does not contain one. When the donor cell and the recipient cell physically touch, the F plasmid is copied over to the recipient, sometimes along with other genetic material in the cell (Russell, 2003). The efficiency of this transfer is low, however, due to the fact that it is hard for the cells to stay touching for very long (Hartwell, 2000). After the F plasmid is copied to the recipient, the recipient cell in now considered a transconjugate cell. It can now take the role of donor cell and copy its genetic material to another recipient. These cells can be plated on different cultures to see if they grow (Prescott, 1999). The cells can then be identified and it can be determined if conjugation has happened successfully. In this experiment, mutant E. coli donor and recipient cells were conjugated and plated on different agar and MacConkey plates to identify the cells, determine whether conjugation was successful, and determine the efficiency of conjugation.
Materials and Methods
Teams were first given culture tubes containing either donor or recipient E. coli cells suspended in broth. The donor cells contained the lac operon on their F plasmid. The recipient cells did not contain the lac operon, but instead had a mutation in a chromosomal gene which made it resistant to the antibiotic chloramphenicol. The cultures were then transferred to plates using the sterile technique, which was used throughout all parts of the experiment. 0.1 ml of a 10-5 and 10-6 dilution of each culture was transferred to separate L-agar plates without chloramphenicol and separate L-agar plates with chloramphenicol. 0.1 ml of a 10-5 dilution of each culture was also transferred to separate MacConkey (MAC) agar plates. The culture was spread by placing small glass balls in the plates to roll around and spread the culture evenly. The small glass balls were removed and the plates were all labeled and incubated overnight at 35 ºC. The next day the number of colonies found in each plate was recorded.
For the next part of the experiment, transconjugate cells were plated and counted. The transconjugate cells were made by pipetting 1 ml of prepared donor cells and 1 ml of prepared recipient cells into an empty sterile tube. This culture was mixed by swirling it. The culture was then incubated for two hours at 35 ºC. Next, a 10-5 and 10-6 dilution of the donor culture were each transferred to separate MAC agar plates without chloramphenicol. 0.1 ml of a 10-5 and 10-6 dilution of the recipient culture were each transferred to separate L-agar plates with chloramphenicol. Finally, 0.1 ml of a 10-4 and 10-5 dilution of the transconjugate culture were each transferred to separate MAC agar plates containing chloramphenicol. The cultures were spread evenly on the plates like before using small glass balls. The plates were all labeled and incubated at 30 ºC overnight. The next day the number of colonies in donor and recipient plates that yielded between 30 and 300 colonies were recorded along with the number purple colonies in the transconjugate plates.
For the last part of the experiment, the plasmids in the donor, recipient, and transconjugate cells were isolated. They were isolated using a QIAprep Spin Miniprep kit and the QIAprep procedure. 1.4 ml of each culture was used for the start of the procedure. The DNA plasmids were each eluted in 50 μl of Tris buffer during the last step. 15 μl of each plasmid DNA was loaded into the gel of the apparatus, and photos of the DNA bands were taken after electrophoresis was finished. The bands were then measured and the points were plotted on semi-logarithmic graph paper.
After one day, the donor cells plated were checked for colonies. The Mac plate with a 10-5 dilution resulted in 37 purple colonies for my group (Table I). The other donor groups in the class counted 24 purple colonies and 103 purple colonies on their Mac plates (Table I). The L-agar plates with chloramphenicol for my group resulted in no colonies for both the 10-5 and 10-6 dilutions (Table I). The other donor groups in the class also found no colonies in their L-agar with chloramphenicol plates (Table I). The L-agar plates free of chloramphenicol for my group resulted with 46 colonies for the 10-5 dilution and 7 colonies for the 10-6 dilution (Table I). The other donor groups found 54 and 46 colonies in their 10-5 dilutions, and 8 and 0 colonies for their 10-6 dilutions, respectively (Table I).
In regard to the groups that plated recipient cells, their Mac plates resulted in 45 white colonies, 40 white colonies, and 31 white colonies (Table I). For their L-agar plates with chloramphenicol, 65 colonies, 30 colonies, and 25 colonies were found for their 10-5 dilutions (Table I). For their 10-6 dilutions, 3 colonies, 4 colonies, and 0 colonies were found, respectively (Table I). Lastly, for the recipient cells plated on L-agar plates without chloramphenicol, 38 colonies, 60 colonies, and 34 colonies were counted from the 10-5 dilutions (Table I). From the 10-6 dilution plates, 1 colony, 5 colonies, and 0 colonies were found, respectively (Table I).
The cells from the second plating were also counted after one night. The donor cells plated on Mac plates resulted in 18 colonies for the 10-5 dilution and 0 colonies for the 10-6 dilution (Table II). The recipient cells plated on Mac plates resulted in 57 colonies for the 10-5 dilution and 3 colonies for the 10-6 dilution (Table II). Lastly, the transconjugate cells plated resulted in 25 purple colonies for the 10-4 dilution and 9 purple colonies for the 10-5 dilution (Table II). Only the counts from the stronger dilutions were used in calculations (Table III).
The original concentration of donor cells plated was found to be 1.8 x 107 cells/ml (Table III). The original concentration of recipient cells plated was found to be 5.7 x 107 cells/ml (Table III). The original concentration of transconjugate cells plated was found to be 2.5 x 106 cells/ml (Table III). The efficiency of recipient cells to transconjugate was 8.77% (Table IV). The average plasmid distance on the gel photo was 52.9 mm and the plasmid size was 2.89 kb.
The donor and recipient cells both grew on the L-agar plates without chloramphenicol. This is because L-agar plates contain simple chemical medium which E. coli can use as its food source (Miller, 1972). Only the recipients grew on the L-agar plates with chloramphenicol, however. Chloramphenicol is an antibiotic that inhibits bacterial protein synthesis (Davies 1994). The recipient cells contained genes on their bacterial chromosome which made them resistant to chloramphenicol. The donor cells did not carry the genetic information to be resistant to chloramphenicol, so they were not able to grow. In regards to the MacConkey plates, the donor cells plated grew into purple colonies while the recipient cells plated grew into white colonies. This is because the donor cells contained an F plasmid that had the lac operon on it. They were considered lac+. The lac operon allows cells to digest lactose, which was on the MacConkey plates. The digestion of lactose produced some acidity. There was an pH indicator in the MacConkey plates that turned the colonies purple in the presence of the acid. This is why the donor cells plated became purple colonies. The recipient cells did not contain an F plasmid, and therefore no lac operon. They were considered lac–. This meant that they could not digest lactose, and thus did not produce the extra acidity to activate the indicator. This is why the recipient cells plated on the MacConkey plates became white colonies.
The transconjugate cells plated on the MacConkey plates produced white colonies and purple colonies. This is because of conjugation between the donor cells and the recipient cells. When the two cells touch, the F factor from the donor cells is copied to the recipient cells (Black, 1999). This means that the transconjugate cells were recipients that contained the F factor, which had the lac operon on it. Not all of the recipient cells in the transconjugate mixture conjugated however, and this is why both white colonies and purple colonies were found. Some of the cells could digest lactose while other could not. If the transconjugated cells were plated on the L-agar plates with chloramphenicol, I would expect them to grow. The F factor from the donors would have been copied to the recipients, and the recipients would still have the genes coding for resistance to chloramphenicol on their bacterial chromosomes. The transconjugates are able to digest lactose, which was tested, and should be resistant to chloramphenicol. The donor cells would still not be resistant to chloramphenicol even in the presence of transconjugate cells because only cells that contain no F plasmid can be conjugated (Hartwell, 2000). The transconjugate cells could not pass the genetic information on to the donor cells because they already would have an F plasmid.
The efficiency of the donor cells to transconjugate the recipient cells was 8.77% (Table IV). This number should be fairly low because of recipients only receive 3% or less of the donor’s DNA (Hartwell, 2000). It is difficult for the cells stay touching together for very long, so the efficiency of conjugation is not going to be very high. The percent found for my group seems larger than average, but I suppose over time more and more of the recipients will conjugate and receive the F factor. Once a recipient is transconjugated, it could become a donor itself, so that would speed the process.
In Tables II and III, the number of colonies found when reducing the concentration from either a 10-5 to 10-6dilution or a 10-4 to 10-5 dilution should have be around a tenth of the colonies found from the stronger concentration. This is because the concentration of cells is reduced by a factor of 10%, and thus only 10% of the original number of cells should be in that concentration. The reason why perfect numbers did not show up is because the sample of cells taken from the mixture and plated could have had more or less cells than normal. The mixture may not have been thoroughly agitated. This is the cause for the error there.
In regards to the gel graph, the donors and transconjugates had plasmids nearly the same size. This is because the donors copy their F plasmid to the recipients, so the transconjugates should have plasmids the same size as the donors. If their plasmids were not the same size, then something probably went wrong during the procedure. This was confirmation that the donors truly did copy their F plasmids to the recipients. The recipients in the gel graph did not show an F plasmid. This is also proof that the recipients really had no F plasmid, and that they received the F plasmid from the donors during conjugation.
Black, Jacquelyn G. 1999. Microbiology: Principles and Explorations (4th Ed). (Prentice Hall, Upper Saddler River, NJ) 786 p.
Davies, Julian. 1994. Antibiotics and Resistance Genes.
Hartwell, L, L. Hood, M.L. Goldberg, A.E. Reynolds, L.M. Silver, R.C. Veres. 2000. Genetics: From Genes to Genomes. (McGraw Hill, Boston) 813 p.
Prescott, L.M., J.P. Marley, and P.A. Klein. 1999. Microbiology (4th Ed). (McGraw Hill, Boston) 962 p.
Russell, Peter J. 2003. Essential iGenetics. (Benjamin Cummings, San Francisco).
|Number of Colonies Found|
|Plate Type:||L-agar without cm||L-agar with cm||Mac|
|Donor 1||54||8||0||0||24 (purple)|
|Donor 2||46||7||0||0||37 (purple)|
|Donor 3||118||0||0||0||103 (purple)|
|Recipient 1||38||1||65||3||45 (white)|
|Recipient 2||60||5||30||4||40 (white)|
|Recipient 3||34||0||25||0||31 (white)|
|Donors on mac||Recipients on mac||Transconjugates on mac|
|Number of Colonies:||18||0||57||3||25 (purple)||9 (purple)|
|Culture||Number of Cells||Volume Plated (ml)||Dilution||Concentration of Original Culture (cells/ml)|
|Donors||18||0.1||10-5||1.8 x 107|
|Recipients||57||0.1||10-5||5.7 x 107|
|Transconjugates||25||0.1||10-4||2.5 x 106|
|Culture||Control of Original Culture (cells/ml)||Total Volume in Original Culture (ml)||Original Number of Cells||Efficiency (original number of transconjugate cells / original number of recipient cells x 100%)|
|Recipients||5.7 x 107||2||2.85 x 107||8.77%|
|Transconjugates||2.5 x 106||1||2.5 x 106|