Aims
By using the characteristics of reporter genes as well as the characteristics of the bacteria themselves, we were to identify four bacteria known originally as A,B,C and D. We had to incorporate the bacteria into plasmid vectors, plate and incubate, then use various methods to properly identify them. After which to ensure that we had successfully recombined the DNA of the bacteria with the plasmid vectors, we had to selective an enzyme which we believed would digest the recombinants into smaller fragments for easier identification.
Other aims were to develop our laboratory skills, such as plating and electrophoresis, also to develop our time keeping and teamwork skills as we worked in pairs to achieve the objectives of each laboratory session. We also aimed to improve our understanding or reporter genes and cloning for the purpose of our respective degrees and the courses we were undertaking.
Method
in order to go about identifying the bacteria we were provided with 4 agar plates, two of which contained the antibiotic Ampicillin at 25µg ml-1 and the other two contained the antibiotic Kanamycin at 50µg ml-1, the plates were divided into quarters and one of the ampicillin plates had 20µl of X-gal and 20µl of IPTG added for the identification of e.coli. Two of the plates were to be stored at 37 degrees and the other two at 30 degrees. Thus the arrangement is as follows:
Later to identify which strain was P.fluorescens we added 1µl of the substrate do-decylaldehyde for the purpose of inducing bioluminescence to the P.fluorescens bacteria in the kanamycin dish incubated at 30 degrees. We also added catechol to this dish in order to identify P.aureofaciens whose colonies turn yellow as a result of cataylsing the conversion of catechol to 2-hydroxymuconic semialdehyde. To identify P.putida both the ampicillin and kanamycin plates stored at 30 degrees were observed in the dark.
For the enzyme digest we used the enzyme EcoRI since all the strains had an EcoRI cutting site which would determine the presence of the bacterial DNA within the recombinant plasmid, the digests then underwent electrophoresis for 45 minutes at 120volts.
Results
The table below shows which unknown strains grew on each plate:
- From this we could identify strain B as being E.coli as it required IPTG to induce the laxZY genes and X-gal to turn the bacterial colonies blue if ϐ-galactosidase was present which in this case it was.
- Strain A was also identified as P.putida as the Amp and Kan dishes stored at 30°C both showed signs of bioluminescence after being viewed in the dark for 10minutes.
- At this stage however C and D could not be identified however D was believed to be P.aureofaciens due to the yellow tinge of the bacterial colonies.
After the addition of catechol, D was confirmed as P.aureofaciens as the yellow colour of the colonies intensified. Addition of do-decylaldehyde however appeared not to induce bioluminescence in C however this was assumed to be P.fluorescens through the process of elimination (the others had been identified).
Below is a table confirming the the results of this part of the experiment:
To confirm this preliminary identification, bacterial samples were inoculated into individual liquid mediums and through a process of centrifuging and and addition of SDS/NaOH and ethanol at various concentrations were purified then suspended in RNase (TE buffer containing 20µg pancreatic RNase ml-1) then had 1µl of the digestive enzyme EcoRI added. After a period of incubation, dye was added and and 10µl of each sample added to a well within the agarose gel, along with samples of a 1Kb DNA ladder and pure samples of the plasmids pUCD607 and pUC18.
From the digest we expected to see that if strains A and B were P.putida and E.coli then fragmentation would occur in their lanes due to there being two EcoRI cut sites in pUCD607 and one cut site in pUC18 However for C and D who were inserted into suicide vectors there would be no fragmentation due to the breakdown of the suicide vectors which would then become incorporated into the bacterial chromosome, thus losing their EcoRI cut sites.
Electrophoresis of the DNA digests of the four bacterial strains
Lane1: 1Kb DNA ladder
Lane2: pUCD607
Lane3: pUC18
Lane4: EcoRI digest of strain A
Lane5: EcoRI digest of strain B
Lane6: EcoRI digest of strain C
Lane7: EcoRI digest of strain D
Lane8: No digest
Note: the bright smudge across the base of the gel is RNA contamination. Also the samples moved towards the positive electrode because DNA is negatively charge.
Fragment sizes as calibrated from DNA ladder:
Lane1: 12,216bp, 7,126bp, 6,108bp, 5,090bp, 4,072bp, 3,054bp, 2,036bp, 1636bp, 1,018bp and 506/517bp (appears as bright couplet amongst smaller fragments)
Lane2: 12,216bp and 6,108bp
Lane3: 4200bp
Lane4: 12,216bp and 6,108
Lane5: 4200bp
Note: the calibration graph can be viewed on the next page
As suggested earlier, the pure plasmid pUCD607 and Strain A both have two fragments confirming that the recombinant does contain P.putida.
The same goes for pUC18 and strain B which features one fragment as a result of one cut site thus confirming the presence of E.coli.
No fragmentation has occurred in lanes 6 and 7 suggesting that the plasmid vectors did breakdown and become incorporated into the chromosomes of P.fluorescens and P.aureofaciens. Though this gel electrophoresis doesn't necessarily confirm the identity of strains C and D, their identities can be confirmed from the earlier results Thus the preliminary identifications are correct.
Discussion
From the results, the experiment went as expected and we were able to identify each of the unknown bacterial strains via either their reporter genes or through the enzyme digest which confirmed the presence of lack of plasmid thus we were able to confirm our preliminary identifications which can be seen again below;
The experiment however was not without problems, such as the agarose gel leaking whilst setting so we ended up with shallower wells than we would have liked. As a result we used the shallowest of the wells to house the digests where we believed no plasmid was present and fortunately this worked. We also had a problem with plate 2 (see diagram in methods) where a couple of the e.coli colonies contaminated the neighbouring quarters, to prevent further contamination we didn't use the other bacteria colonies on this plate for later parts of the experiment.
To further improve on our identification methods, the following experiments could have been undertaken;
Phage typing: Different types of bacteria respond differently in the presence of viral phages. To run this experiment we could have plated the bacteria as before (each plate divided into quarters) then to each plate added a phage that only infected a particular bacterial species. After incubation, where bacterial growth failed would represent which species was susceptible to the viral phage used on that plate.
Protein Analysis: Each bacteria has its own set of proteins, through a combination of western blotting to identify particular proteins unique to each bacterial species and electrophoresis to break down the proteins by mass, it would be possible to identify each bacterial species by the proteins they express.
Agglutination test: This method is used to identify bacterial antigen and in turn the bacteria that produce them. In this test, we place a sample of unknown bacteria in a saline solution and mix with an antigen that is specific to a particular species/strain of bacteria, if the antigen causes the unknown bacteria to agglutinate then the test will identify the bacteria as being identical to that for which the antigen was produced against. This test is particularly using for identifying strains within a species. This method of experimentation could however be considered very similar to that of phage typing.
Sequence analysis: We could undergo sequence analysis of the bacterial strains (via PCR or as would be easier and cheaper in our case, by using the known sequences of the plasmids used) then compare them to the known DNA sequences of each species to order to get a match and positively identify each bacteria.
All the above methods could be used to give a more thorough identification of strains C and D since the plating stage had be relied on in our case in order to identify them. The Electrophoresis could not be relied on for identification except to confirm the breakdown of the plasmid by its lack or presence as fragments after enzyme digest. However to conduct these experiments would also require an increased availability of time and resources, plus costs have to be taken into consideration. However they should be considered for future attempts of bacterial identification.
MB3005
Molecular Biology of the Cell
Identification of Bacteria containing either plasmid-borne or chromosomal reporter genes
07th December 2008