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Each of the graphs follow distinct growth phases of the E.coli K12, 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 graph, we see a late log phase where ther 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 consant due to nutrients starting to become a limiting factor on the growth of the bacteris, 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 conatiing 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. Primarly 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 requeied energy soruces 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 gylerol medium and also as the growth eneters the stationary phase, there are bacteruial 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 sunbstare of lactose and so the cell does not transcribe the gens to make this enzyme as it would be a waste of energy. It would be expected that the assay would show no levels of of this enzyme present however.................
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 allolactose, which binds to an allosteric site on the repressor protein and consequently resulting in a conformational shape change which means that the repressor protin is nto 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 bing 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 teh genes being transcribed as so it is said to be polycistronic, this then enables translation of the polypeptide B-galactosidase to that thisenzyme can metabolise the lactose into glucose and galactose, this causes the call to become more permeable to this substare 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 rults in the calls being able to double rapidly on the sunstrate 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 expreesed 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 engey conservation.
Th edobling time of glucose obtained form 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 se more than one type of sugar as an engery 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 enxymes to metabolise glucose are already avalible 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 dobling 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 absorbace 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 continuos 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 futher into the transcription an 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 whichnear to the lac operon at a CAP site, nar 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 achived when the lactose is present and glucose is absent. When glucose is present it causes the cAMP levels to becme 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 eail 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 tha the bacteria aree able to metabolise two different nutrient supplies. Also the E.coli adopts a mechanism which eables it to utilise the carbon soruce which can be meatbolised 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 ahave been activates they sre able 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 drwn graphs and calculations and also the computer program excel. When obtaining these values from the handdrawn graphs there are many soruces 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 anf 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 accuate representation of the data and so he results for the calculations are moreaccut=rate. It is largely evident how much more accurate the results are form the excel calculations and graphs because the tobling 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 dobling time with the control flask. The exce graph shows an order of the quickest being the glucose and lactose, lactose, gucose 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 ablt to use a variety of nutrient resources to obtain enegery whilst also maining the more energy effienect way of doing so which is why it is able to adapt and to survive and compete against other microorganisms in harsh environments.