Escherichia coli are gram negative bacteria which are mainly resident in the gut of mammals were it has to stand the harsh environment and be able to obtain maximum amount of energy when at times there may be limited nutrient sources available; In order to achieve this E.coli have developed a mechanism which allows them to control the expression of certain gens which in turn controls what enzymes are produced so that the only enzymes which are produced are the ones that are needed which consequently leads to efficiency of energy usage.
The lac operon is a cluster of genes which encodes for three enzymes: B- galactosidase, galactosidase permease and galactosidase acetylase. These enzymes are required to metabolise lactose and so E.coli only induces the transcription of these enzymes when lactose is available. When lactose becomes available, the lac genes on the lac operon are expressed by allolactose being formed and binding to a lac repressor protein which prevents the lac repressor protein from binding to the operator which allows RNA polymerase to attach and bind to the promoter and consequently transcribe the genes so that the enzymes can be translated. Absence of the repressor is needed for transcription to occur but another DNA binding protein is also required, this being catabolite activator protein or CAP, which binds to Cyclic AMP and then binds to the DNA in order to assist RNA polymerase and hence for transcription to occur
The aim of this investigation was to look at how different nutritional factors, glucose and lactose, affect the growth of E.coli K12 (lac+) and hence how much B-galactosidase is produced. This will show hoe the E.coli uses this mechanism in order to conserve energy and also to see how the production of the enzyme alters when just glucose is present, just lactose is present, both glucose and lactose are present an finally when neither are present and hence investigate how and what nutrients induce the polycistronic gene of the lac operon
Four flasks, containing 45ml o.2% mineral glycerol medium, were prepared. 2.0 ml of sterile water was added to one flask, which was the control, another flask with 1.0ml of both sterile water and lactose (10%), labelled the Lac flask, another with 1.0ml of both water and glucose (10%), labelled the Glu flask and finally 1ml of both glucose (10%) and lactose (10%) to the fourth flask, labelled Lac and Glu flask.
2.0ml of bacterial suspension, E.coli K12 (lac+) culture, was then added to each flask and measured spectroscopically at 540nm.
The four flasks were then incubated in a shaking incubator at 37oC and samples were then taken every 30 minutes for 6 hours and measured using the spectrophotometer at 540nm.
Method for B-Galactosidase Activity
In a fume cupboard 1.0ml of E.coli K12 (lac+), 1 drop of toluene (UNITS) and 1.0ml ONPG (orthnitrophenyl B-D-galactoside) was added to a test tube and mixed well, This was then incubated for 10 minutes at 35o C. 0.5ml of 1M sodium carbonate was then added and mixed. This was then measured spectroscopically at 420nm, this test was repeated at the end of the day.
Method for Gram staining
A drop of culture was placed onto a microscope slide and left to evaporate, the bacterial cells were then fixed onto the slide by passing it through a Bunsen burner. Crystal violet was then added for one minute and then washed off and flooded with logol's iodine for one minute; this was then washed off with water and then dripped with alcohol (ethanol) for 10 seconds and then washed off immediately. Safarnin was added for two minutes and removed with tap water, the slide was then left to dry
The absorbance, at 540nm and the natural log of the absorbance values of the fours flasks, over six hours, is presented in table one. Four growth curves were plotted for the natural log of absorbance against time (minutes) using the values from the table, for each of the different flasks containing different nutritional factors (see graphs 1,2,3 and 4).
From the growth curves, the exponential phase was observed and the absorbance readings for the time values were converted into biomass by using a standard curve which relates A540 to dry weight in ug mL-1 for E.coli cells. The natural log of the biomass values was then calculated to give dry biomass for each of the four flasks (shown in table 2,3,4 and 5) and plotted against time (in hours), presenting the exponential phase of microbial growth in dry biomass (see graphs 5, 6, 7 and 8).
The effect of different Nutritional Factors on the growth of E.coli K12 (lac+)
The Dry Biomass of E.coli K12 (lac+) of the exponential phase when there is no Glucose or Lactose present (The Control)
To 3 d.p u?
(Ln) Dry Biomass
Exponential phase values for Dry Biomass of E.coli K12 (lac+) when Glucose is present
(ln) Dry Biomass
To 3 d.p
Exponential phase values for Dry Biomass of E.coli K12 (lac+) when Lactose is present
To 3 d.p
(Ln) Dry Biomass
To 3 d.p
The Values from the Exponential growth phase converted to Dry Biomass for E.coli K12 (lac+) when both Lactose and Glucose is present
To 3 d.p
(Ln) Dry Biomass
To 3 d.p
Assay for B-galactosidase for each bacterial culture
Nutritional factors of the culture mediums
Absorbance at 420nm
Normalisation of results to 3 d.p
After 1.5 hours
After 4.5 hours
(absorbance after 4.5 hr) * td
Control (no glucose or lactose)
Lactose and Glucose
Table 6 shows how a the computer package (Excel) was used to calculate the doubling time for E.coli, using the graphs 9,10,11 and 12 ; from these values calculations were made as to how many organisms were present in each culture at the time the B-galactosidase assay was conducted so that the enzyme assay results could be normalised.
Table 7 shows the contamination percentage of each culture, from other gram positive microorganisms other than E.coli K12 (lac+), such as normal flora from skin, so that considerations could be made as to whether any metabolic activity was from E.coli alone or were any other microorganisms involved.
Contamination (%) of different culture mediums using a Gram Stain
Culture Medium containing E.coli K!2 (lac+) and other nutritional factors:
Starter seed Culture
No glucose or Lactose- Control
Both Glucose and Lactose
Using graphs 5,6,7 and 8 I was able to work out the growth rate constant (U)for E.coli K12 (lac+)using the equation (lnXt-lnX0)/(t-t0) and substituting the values form the graph into this equation. From the values obtained for the growth constant, the doubling time was then calculated (td):
The control- no glucose or lactose present:
= 0.894 h
To convert to minutes: 0.8948* 60
Doubling time (td)= 53.65 minutes to 2d.p
= 0.776 h
To convert to minutes: 0.776* 60
Doubling time (td)= 46.56 minutes to 2 d.p
= 0.748 h
To convert to minutes: 0.748* 60
Doubling time (td)= 44.88 minutes to 2 d.p
Glucose and Lactose Present
= 0.846 h
To convert to minutes: 0.846* 60
Doubling time (td)= 50.76 minutes to 2 d.p
The computer package (excel) was then used to construct the same graphs as 5,6,7 and 8, however using excel to give a more accurate line of best fit so that more accurate values for doubling time (td) could be calculated. Graphs 9,10,11 and 12 represent this:
Calculations For Excel Graphs
Control- No glucose or lactose Present:
td =52.90 minutes to 2 d.p
td =49.42 minutes to 2 d.p
td =48.42 minutes to 2 d.p
Glucose and Lactose Present
td =43.40 minutes to 2 d.p
Each of the graphs follow distinct growth phases of the E.coli K12 _(lac+), the first two point of each graph indicate a lag phase, which is the time it takes for the bacteria to become fully metabolically active to allow time for biosynthesise to take affect; consequently the first two points at time 0.0 minutes and 30.0inutes were not included in graphs 5-12 as these graphs are measuring the phase at which the bacteria are fully metabolically active when enzymes may be produced.
The next phase shown on the growth curves, graphs: 1,2,3,4 is the exponential phase from the points taken at 60 minutes up to 150 minutes, which mimics a first order chemical reaction; this is when the E.coli are producing many primary metabolites such as the enzyme B-galactosidase. The bacterial cells are metabolically active and doubling at constant rate with the prime focus being to increase cell mass. Towards the end of the exponential phase between 150 and 180 minutes, on each of the four graphs, we see a late log phase where there is a gradual move across into the stationary phase, this may be due to a lack of nutrients or the build up of waste product from the E.coli. Each graph shows the transition into the stationary phase where the cell count of the bacteria is starting to level off and become constant due to nutrients starting to become a limiting factor on the growth of the bacteria, it is at this stage when secondary metabolites may be produced. Graph one for the control culture medium, containing no glucose or lactose, reaches the stationary phase slightly quicker, at around 180 minutes, at a lower natural log of absorbance( -3.5) than the rest of the cultures, this is most likely due to this culture not containing any nutrients and so the cells mass does not reach as a high a value as bacteria who are supplied with nutrients, such as glucose and lactose, which the bacteria can metabolise and provide energy for cell division.
The time span that the inoculated samples were measured at does not represent the full phases of microbial growth as each of the graphs 1,2,3 and 4 do not display the death phase where cell numbers start to decrease, this indicates that for a repeated procedure of the experiment samples should be taken over a greater time span of more than 6 hours.
The flask witch served as the control for the experiment, as it had no glucose or lactose present, had a low doubling, and a low production of b-galactosidase in comparison to the rest to the other culture mediums. Primarily this is because the bacteria had no nutrients to metabolise into energy, which is required for the bacterial cells to divide by binary fission and so without the required energy sources the growth of the E.coli will be limited. However it needs to be pointed out that there were still some growth within this flask as the E.coli will be able to obtain energy from the glycerol medium and also as the growth enters the stationary phase, there are bacterial cells dyeing as well as some multiplying, this is made possible as when some of the cells die they release nucleic acids and peptides which provide energy for the other multiplying cells.
The control shows a reduced enzyme assay result for the B-galactosidase as there is not lactose present for the lac operon to switched on and so the this enzyme is not needed to break down the specific substrate of lactose and so the cell does not transcribe the gens to make this enzyme as it would be a waste of energy. Despite this there is a limited amount of protein production as there is elevated levels of cAMP and CAP which bind together and bind to the DNA and therefore assist RNA polymerase which increase expression of the genes however this is limited because along with is the repression expression due to the binding of the lactose repressor with the promoter.
The flask containing Lactose has a very high enzyme assay result for B galactosidase because this enzyme is induced in the presence of lactose. This is because when lactose is present some of the lactose molecules is converted into all lactose, which binds to an allosteric site on the repressor protein and consequently resulting in a conformational shape change which means that the repressor protein is no longer complementary to the shape of the lac operator and so cannot bind to it. The repressor protein and hen no longer prevent the operon genes from being transcribed; RNA Polymerase is then able to bring to the operator and transcribe the lac genes, such as the LAC I gene which translate to the enzyme b-galactosidase. When the lac genes are transcribed, mRNA is produced which carries the information for all the genes being transcribed as so it is said to be polycistronic, this then enables translation of the polypeptide B-galactosidase to that this enzyme can metabolise the lactose into glucose and galactose, this causes the call to become more permeable to this substrate and so obtain the nutrients it needs to increase in cell mass; This mechanism is only switched on when lactose is present to conserve energy and results in the calls being able to double rapidly on the substrate lactose and hence why lactose has a quick doubling time as it switches on this mechanism to rapidly utilise the lactose nutrient to provide energy for Binary Fision.
The Flask containing Glucose has a very low absorbance result for enzyme assay of B-galactosidase. This because when lactose is not present the lac genes are note expressed to ensure then that the E.coli uses its energy efficiently. This is achieved via negative regulation by the repressor protein as it binds to the RNA polymerase and hence prevents transcription of the lac genes as the RNA cannot be synthesised without RNA polymerase and so the lac operon is down regulated. This is very valuable adaptation of the E. Coli K12 (lac+) in terms of energy conservation.
The doubling time of glucose obtained from excel is 49.42 minutes which is very similar to the doubling time obtained for lactose which was 48.42 minutes, this shows that E.coli is able to catabolise both nutrients equally to obtain energy which is an advantage as it is able to see more than one type of sugar as an energy source giving it more versatility to survive in different environments; The only difference is that the enzymes needed to break down glucose are already available to the bacterial cell whereas with lactose to the enzymes required have to be translated from the induction of the lac genes to be transcribed. The doubling time of the E.coli within the lactose flask had a slightly quicker doubling time than the glucose flask, which is unexpected as like said previously the enzymes to metabolise glucose are already available however it is important to consider that this flask showed 5 % of contamination from the gram stain and so this increase in doubling time may be as a result of some gram positive bacteria which are doubling in mass at a quick rate and hence slightly increasing the doubling time for lactose over that of glucose.
The Flask that contains both glucose and lactose has a fairly high enzyme assay result of 1.300 absorbance at 420nm, however not as high as the results obtained for lactose which gave an absorbance of 2.080 at 420nm, due to the fact that the genes needed for lactose metabolism are only transcribed at a limited rate. This is because glucose is a better carbon source for E.coli as it only takes two enzymes to metabolise it, which are readily available and by continuous translation of these enzymes, and E.coli will use up glucose before it uses the lactose nutrient source since glucose is the most commonly available nutrient for E.coli. Looking further into the transcription and translation processes of the E.coli it can be seen that glucose affects the concentration of cyclic AMP (which derived from ATP) as glucose is inversely proportional to cyclic AMP which means that as glucose decreases the concentration of Cyclic AMP increases. The presence of catabolite activator protein (CAP) influences the activity of RNA polymerase which is needed to transcribe the lac genes. cAMP binds to CAP which near to the lac operon at a CAP site, near the promoter and aids in the attachment of RNA polymerase so transcription can proceed, however this binding of the cAMp and CAP can only be achieved when the lactose is present and glucose is absent. When glucose is present it causes the cAMP levels to became so low that they cannot bind to CAP and hence cannot bind to the DNA and RNA polymerase is not assisted and consequently transcription of the lac genes cannot proceed. This explains why glucose is used up first as while ever glucose is present the lac genes cannot be transcribed and so the required proteins to metabolise lactose are not being translated and so lactose is not used, however was all the glucose has been used up catbolite repression is abolished, cAMP levels rise and are able to bind to CAP and so the lac operon is expressed and the bacteria are then able to metabolise the lactose. This is a good advantage to the E.coli as it ensures that the bacteria use the carbon source which is more eailsly metabolised first which again conserves energy.
The doubling time for the flak that contains both glucose and lactose is also quicker than the rest of the flasks as it has a doubling time of 43.40 minutes This is most likely due to the fact the the bacteria are able to metabolise two different nutrient supplies. Also the E.coli adopts a mechanism which enables it to utilise the carbon source which can be metabolised most easily first before switching of the lac operator to produce the enzymes such as B-galactosidae to break down lactose also once these enzymes have been activates they sure able to metabolise the lactose rapidly to metabolise the lactose rapidly.
The specific growth rate (u) is the generation time of a culture growing exponentially, from this the doubling time (td) which is the time it takes for the bacteria to double in size, can be calculated. This was done using hand drawn graphs and calculations and also the computer program excel. When obtaining these values from the hand drawn graphs there are many sources of error, such as the interpretation of where the line of best fit should be, also where the points lie with reference to the axis and finally when reading values of the graphs it is hard to visualize past 2 decimal places as so the values are not as precise as they could be. Using excel allows an exact line of best fit to be used which is an accurate representation of the data and so he results for the calculations are more accurate. It is largely evident how much more accurate the results are form the excel calculations and graphs because the doubling times are in a different order for the different cultures using the two different methods: The hand drawn graphs and calculations give an order, starting with the quickest of lactose flask, glucose flak, glucose and lactose flask and finally the slowest doubling time with the control flask. The excel graph shows an order of the quickest being the glucose and lactose, lactose, glucose and finally the slowest being the control. This presents just how important accuracy is as it can completely change the evaluation of the results.
Overall is clearly evident that the E.coli is a remarkable microorganism which is able to use a variety of nutrient resources to obtain energy whilst also maintaining the more energy efficient way of doing so which is why it is able to adapt and to survive and compete against other microorganisms in harsh environments.