Biosensor Characterisation

From 2006.igem.org

(Difference between revisions)
Jump to: navigation, search
Line 214: Line 214:
The composition of the solutions used in this experiment are the same as those used in Experiment 6 except the culture used is Biobrick construct 2 rather than 1.
The composition of the solutions used in this experiment are the same as those used in Experiment 6 except the culture used is Biobrick construct 2 rather than 1.
 +
It looks like 7B is an anomolous reading as none of the other experiments led to an increase in pH.  The pH decreases from the start and continues to decrease over time.  This indicates that the bacteria are breaking down the lactose to acid which shows that the LacZ part of this Biobrick construct is functioning correctly.  However, the results above show that the concentration of arsenic present does not affect the response of BioBrick construct 2 under the conditions shown in the table.  This could mean that either the arsenic responsive part is not functioning correctly or else all of the concentrations of arsenic tried in this experiment are too low to induce a response different to that of the control. 
 +
 +
 +
==Experiment 8 - Effect of arsenic and lactose concentration on rate of pH change of arsenic responsive E-Coli (Biobrick construct 2) - Repeat==
 +
 +
Experiment 8 was carried out using Biobrick construct 2 which has an arsenic promoter coupled to a LacZ.  Different concentrations of arsenic and lactose were added to the solution to see which concentration produced a response. 
 +
 +
{| border="1"
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
|-
 +
| '''Experiment:''' || '''Control''' || '''8B''' || '''8C''' || '''8D'''
 +
|-
 +
| '''Arsenic (ppb):''' || 0  || 500 || 5000 || 0
 +
|-
 +
|-'''Lactose (ml):''' || 2.5 || 2.5 || 2.5 || 0
 +
|}
[[Image:Expt_8_Graph.JPG | Experiment 8 Graph | 800 px]]
[[Image:Expt_8_Graph.JPG | Experiment 8 Graph | 800 px]]
 +
 +
{| border="1"
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
!width="100"|
 +
|-
 +
| '''Experiment:''' || '''Control''' || '''8B''' || '''8C''' || '''8D'''
 +
|-
 +
| '''LB (ml):''' || 50 || 50 || 50 || 50
 +
|-
 +
| '''Culture (ml):''' || 5 || 5 || 5 || 5
 +
|-
 +
| '''Lactose (ml):''' || 2.5 || 2.5 || 2.5 || 0
 +
|-
 +
| '''Ampicillin (μl):''' || 150 || 150 || 150 || 150 
 +
|-
 +
| '''Arsenic (ml):''' || 0  || 0.33 || 3.3 || 0
 +
|-
 +
| '''Total volume (ml):''' || '''57.65''' || '''57.98''' || '''60.95''' || '''55.15'''
 +
|}
[http://2006.igem.org/University_of_Edinburgh_2006 Main page]
[http://2006.igem.org/University_of_Edinburgh_2006 Main page]

Revision as of 14:22, 14 August 2006

Contents

Experiment 1 - Effect of solution volume on rate of pH response

Experiment 1 was carried out using IPTG responsive E-Coli with LacZ to find out what difference the total solution volume makes to the rate of pH change.

Experiment: Control 1B 1C 1D
Solution volume c.f control: 100% 50% 20% 10%

Experiment 1 Graph


Experiment: Control 1B 1C 1D
LB (ml): 50 25 10 5
Culture (ml): 5 3.125 1.25 0.625
Lactose (ml): 1.25 0.781 0.313 0.156
Ampicillin (μl): 10 6.25 2.5 1.25
IPTG (μl): 10 6.25 2.5 1.25
Total volume (ml): 56.27 28.919 11.568 5.784


The solution with total volume 28.1375 ml (1B) produced the fastest initial rate of pH decrease and reached approximately the same final pH as the control (pH 4.84).

The smallest solution volume changed pH at the slowest rate and after an initial decrease in pH, it then increased to 7.87 overnight.

Therefore, in the optimum order, 1B > Control > 1C > 1D


Experiment 2 - Effect of solution dilution on rate of pH response

Experiment 2 was carried out using IPTG responsive E-Coli with LacZ to find out what difference the volume of sterile water added to the solution makes to the rate of pH change.

Experiment: Control 2A 2B 2C
Sterile water (ml): 0 50 25 10

Experiment 2 Graph


Experiment: Control 2A 2B 2C
LB (ml): 50 50 50 50
Culture (ml): 5 5 5 5
Lactose (ml): 1.25 1.25 1.25 1.25
Ampicillin (μl): 10 10 10 10
IPTG (μl): 10 10 10 10
Sterile water (ml): 0 50 25 10
Total volume (ml): 56.27 106.27 81.27 66.27


It can be seen that all the dilutions behave in roughly the same way, especially when the error in pH of +/- 0.2 is taken into account. The aim of this experiment was to find out whether it is feasible to dilute the standard culture solution by 50%. The pH of solution 2A which was diluted by 50% decreased at a rate very similar to that of the control solution, therefore it will be feasible to use this dilution in the final biosensor.


Experiment 3 - Effect of shaking on rate of pH response

Experiment 3 was carried out using IPTG responsive E-Coli with LacZ to find out what effect shaking has on the rate of pH change.

Experiment: Control 3A 3B
Shaken: Yes No No

Experiment 3 Graph


The composition of solutions 3A and 3B is the same as that of the control (which is the identical to the control composition used in experiment 1). The only difference in the experiment is that neither solution 3A or 3B is shaken whereas the control is.

It can be seen that for the first 4 hours, the pH of the unshaken cultures decreases at a faster rate than that of the shaken control culture. However, the pH of the unshaken cultures stops falling after approximately 5 hours and begins to increase again. This shows that for the initial experimental period, it is not necessary to shake the biosensor but following the first 5 hours (approximately), shaking becomes more important.


Experiment 4 - Effect of air volume on rate of pH response

Experiment 4 was conducted using different conical flask sizes to find out what effect the volume of air present has on the rate of pH change of IPTG responsive E-Coli with LacZ.

Experiment: Control 4A 4B
Conical flask size (ml): 250 50 100

Experiment 4 Graph

The composition and volume of 4A, 4B and the control solution is that used for the control in Experiment 1 above. The only difference in the experiment is the conical flask volume. The control is a 250 ml flask, 4A is a 50 ml flask and 4B is a 100 ml flask.

In the first 4 hours, the flask containing the smallest volume of air responded at the fastest rate. After about 5 hours, both 4A and 4B seemed to plateau out at a final pH just above 5 whilst the control continued to decrease slightly. However, if an error in pH of +/- 0.2 is considered, it can be seen there is not much difference in the rate of response between the flasks containing different air volumes.


Experiment 5 - Effect of freezing on pH universal indicator

This experiment was planned but not actually carried out as it became apparent that it would be better to freeze-dry the bacteria and nutrients rather than freezing them at -80 °C.


Experiment 6 - Effect of arsenic concentration on rate of pH change of arsenic responsive E-Coli (Biobrick construct 1)

Experiment 6 was carried out using Biobrick construct 1 which has an arsenic promoter coupled to a LacZ. Different concentrations of arsenic were added to the solution to see which concentration produced a response.

Experiment: Control 6B 6C 6D 6E 6F
Arsenic (ppb): 0 5 10 20 50 500

Experiment 6 Graph

Experiment: Control 6B 6C 6D 6E 6F
LB (ml): 50 50 50 50 50 50
Culture (ml): 5 5 5 5 5 5
Lactose (ml): 1.25 1.25 1.25 1.25 1.25 1.25
Ampicillin (μl): 10 10 10 10 10 10
Arsenic (μl): 0 3.3 6.6 13.2 33 330
Total volume (ml): 56.273 56.274 56.277 56.283 56.303 56.600

The results above show that the concentration of arsenic present does not affect the response of BioBrick construct 1 under the conditions shown in the table. Initially there is a decrease in pH in all tests but we believe this is due to the bacteria metabolising nutrients present in the LB medium to produce an acidic waste product. Then once this nutrient is consumed, the bacteria begin to break down amino acids, also present in the medium and this leads to the subsequent increase in pH. The fact that the pH only decreases slightly at the start indicates that they are not breaking down the lactose to acid which shows that this Biobrick construct is not functioning correctly. It was thought that this would be the case with one of the constructs so this is a useful result.


Experiment 7 - Effect of arsenic concentration on rate of pH change of arsenic responsive E-Coli (Biobrick construct 2)

Experiment 7 was carried out using Biobrick construct 2 which has an arsenic promoter coupled to a LacZ. Different concentrations of arsenic were added to the solution to see which concentration produced a response.

Experiment: Control 7B 7C 7D 7E 7F
Arsenic (ppb): 0 5 10 20 50 500

Experiment 7 Graph

The composition of the solutions used in this experiment are the same as those used in Experiment 6 except the culture used is Biobrick construct 2 rather than 1.

It looks like 7B is an anomolous reading as none of the other experiments led to an increase in pH. The pH decreases from the start and continues to decrease over time. This indicates that the bacteria are breaking down the lactose to acid which shows that the LacZ part of this Biobrick construct is functioning correctly. However, the results above show that the concentration of arsenic present does not affect the response of BioBrick construct 2 under the conditions shown in the table. This could mean that either the arsenic responsive part is not functioning correctly or else all of the concentrations of arsenic tried in this experiment are too low to induce a response different to that of the control.


Experiment 8 - Effect of arsenic and lactose concentration on rate of pH change of arsenic responsive E-Coli (Biobrick construct 2) - Repeat

Experiment 8 was carried out using Biobrick construct 2 which has an arsenic promoter coupled to a LacZ. Different concentrations of arsenic and lactose were added to the solution to see which concentration produced a response.

Experiment: Control 8B 8C 8D
Arsenic (ppb): 0 500 5000 0

Experiment 8 Graph

Experiment: Control 8B 8C 8D
LB (ml): 50 50 50 50
Culture (ml): 5 5 5 5
Lactose (ml): 2.5 2.5 2.5 0
Ampicillin (μl): 150 150 150 150
Arsenic (ml): 0 0.33 3.3 0
Total volume (ml): 57.65 57.98 60.95 55.15


[http://2006.igem.org/University_of_Edinburgh_2006 Main page]

Retrieved from "http://2006.igem.org/wiki/index.php/Biosensor_Characterisation"
Personal tools
Past/present/future years