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Sadam Raziyev(sr13771)
Investigation of the Escherichia coli Lac Operon.
The date of experiment is 31 of October 2013
The purpose of the experiment was firstly to measure beta-galactosidase activity, the ability to ferment lactose in a group of E.coli and secondly to use this data to conclude which of the strains has a wild-type lac operon, which has mutation in lacZ, lacY or lacI.
For methods refer to practical protocol.
E. coli bacteria species contain many genes that are expressed constitutively, which simply means they are always switched on. However, some genes are expressed only when their products are required by the cell. The good example is Lac Operon. This is an operon or set of genes which is responsible for metabolizing and transporting of lactose molecules. Normally E.coli use glucose as the main source of energy as it is smaller molecule and so easily to break down. However, Lac Operon provides a bacteria an opportunity to use lactose when no glucose is present. This increases the chances of bacteria living in gut to survive.
Lac Operon contains promoter region, operator site and sequence of 3 genes which are transcribed to single mRNA. The first gene, lacZ, encodes for beta-galactosidase(LacZ). This is an enzyme which converts lactose to allolactose, and also plays role in lactose hydrolysis.( products are glucose and galactose). The next gene, lacY produces beta-galactosidase permease when expressed. This protein is involved in lactose passage through cell membrane.
The function and role of last gene, lacA is not known yet.
Right next to Lac Operon promoter region another gene can be found. This is a lacI gene, which encodes for repression protein, LacI. Normally when lactose is absent gene lacI is expressed, this results in LacI binding to promoter region of Lac Operon and therefore RNA polymerase can not bind, hence no transcription occurs.
However, when lactose in absorbed beta- galactosidase converts it into allolactase (despite repression of Lac Operon some of an enzyme is still present in bacteria) this then binds to LacI and the result is breaking of bonds between repressor and an operator site. Therefore, transcription occurs and gene is expressed.
In addition, further transcription can be altered by CAP binding. It is activated by the binding with cAMP. The amount of cAMP increases in the absent of glucose. CRP binds to activator site upstream Lac operon promoter region and help the RNA polymerase to attach to DNA stronger. Hence, better transcription. b
Bacteria as all other organisms constantly mutate. Sometimes these mutations occur in Lac operon. This can be fatal to bacteria if the mutation results in malfunction of an operon. To see which mutations are more or less harmful this experiment was performed. 2 experiments produced some results, which are discussed in the next sections.
Results:
The experiment used to compare activity of an enzyme in 4 different E.coli sub-cultures namely UB121, UB122, UB123 and C600. One group of bacteria species were treated with IPTG( mimic of allolactase which cant be hydrolyzed by beta-galactosidase) while another one wasn’t. The difference in behavior were then recorded in tables. The second experiment was used to see which strains of E.coli are able to ferment lactose on McConkey agar plate.
Experiment I.
Table 1. Shows the data for strains grown in absence of IPTG
|
Sub-culture |
Absorbance at 420 nm |
|||||
|
Reading 1 |
Time taken |
Reading 2 |
Time taken |
Reading 3 |
Time taken |
|
|
UB121 |
0.112 |
0 |
0.049 |
5 min |
0.057 |
10 min |
|
UB122 |
0.178 |
0 |
2.570 |
5 min |
2.8 |
10 min |
|
UB123 |
0.150 |
0 |
0.065 |
5 min |
0.070 |
10 min |
|
C600 |
0.207 |
0 |
0.087 |
5 min |
0.091 |
10 min |
The time period was 5 minutes between each reading. This was done because using time period of 30 secs didn’t give reliable results.
Table 2. Shows the results for strains grown in absence of IPTG
|
Sub culture |
Change in absorbance per minute |
OD at 600 nm |
Relative enzyme activity |
|
UB121 |
1.6e-3 |
0.298 |
5.34e-3 |
|
UB122 |
0.046 |
0.534 |
0.086 |
|
UB123 |
1e-3 |
0.344 |
0.003 |
|
C600 |
8e-4 |
0.382 |
2.09e-3 |
OD (optical density) at 600 nm was used because at this wavelength bacteria don’t absorb much light. Also relative enzyme activity, which is the Absorbance:OD ratio, was used here, as it is proportional to the number of enzyme present.
Table 3. Shows the results for strains grown in presence of IPTG
|
Sub-culture |
Change in absorbance per minute |
OD at 600 nm |
Relative enzyme activity |
|
UB121 |
6.2e-3 |
0.285 |
0.022 |
|
UB122 |
8.2e-3 |
0.312 |
0.0216 |
|
UB123 |
0.000003= 0.0 |
.0.336 |
0 |
|
C600 |
0.0746 |
0.341 |
0.219 |
Experiment II.
Table 1. Shows the presence or absence of lactose fermentation in bacteria sub-cultures in MacConkey plate.
|
Sub- culture |
Lactose fermentation (Y/N) |
|
UB121 |
Yes |
|
UB122 |
Yes |
|
UB123 |
No |
|
C600 |
No |
Table 2. Shows activity of an enzyme
|
Activity (Y/N) |
Wild type |
lacZ mutation |
lacI mutation |
lacY mutation |
|
Induced |
Yes |
No |
Constant |
Yes |
|
MacConkey |
Yes |
No |
Yes |
No |
Here induced means the strains of bacteria, which were treated with IPTG and MacConkey, refers to plate where 4 strains were growing. These data provides idea that two strains are able to ferment lactose while two others cannot.
Discussion:
First predictions
Wild type strain is seems to be UB121. According to Table 1. In experiment II these bacteria are able to ferment lactose, which means their Lac Operon, is working normally, therefore activity is present on MacConkey plate. Additionally, activity of beta-galactosidase is present in Induced growth (presence of IPTG) which suggests that no mutations occur or mutations do not cause any effect of enzyme activity. Also, by comparing table 2 and table 3( Exp I) we can see that very little activity is present in U conditions ( not induced) while normal activity is seen in I ( induced ) conditions. All these suggest that UB121 can ferment lactose when it is present, they also can ferment it with the addition of IPTG. However, when none of these is present they activity drops dramatically.
Data for UB122 strain shows clear difference among other strains. According to Exp I these bacteria are able to ferment lactose in both Induced and non-Induced conditions. This may suggest that Lac operon cannot be repressed. So UB122 seems to have lacI mutation. This mutation encodes for abnormal repressor protein which therefore cant bind to operator site and hence transcription is always switched on even in the absence of IPTG. The second Experiment shows that UB122 show beta-galactosidase activity in the presence of lactose. This definitely means that there are no problems with Lac Operon but lacI.
UB123 linked to lacZ mutation. Both experiments show malfunction of Lac Operon as no activity is present in both cases. In experiment I relative activity equals almost 0, this means no beta-galactosidase is present or its function is lost. Comparing with the second experiment where no activity was seen in lactose rich environment lead to the conclusion that UB123 has a faulty lacZ gene.
The last mutation which is the lacY mutation is linked to C600 strain. There is no activity in MacConkey plate test which clearly states that Lac Operon doesn’t function properly. Furthermore, high activity is present in Induced conditions. This is explained by the fact that IPTG doesn’t require beta-galactosidase permease to enter the cell. So in the presence of IPTG lac operon can still function properly regardless of the lacY gene mutation.
Conclusion
W.type lacZ lacI lacY
UB121 UB123 UB122 C600
No unexpected results were obtained. Both experiments show clear difference between strains.