From 2010.igem.org

Arginine is an amino acid that is positively charged. Therefore if B. subtilis 168 is able to take up arginine, it will cause a pH change in the media. This would result in an increase in pH.

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Figure 1: Arginine test using pH indicator stick to measure pH changes of the media.

Time (in minutes)	Control (1)	Control (2)	Control (3)	Test (1)	Test (2)
0	pH 7	pH 7	pH 7	pH 7	pH 7
30	pH 7	pH 7	pH 7	pH 7	pH 7
60	pH 7	pH 7	pH 7	pH 7	pH 7
90	pH 7	pH 7	pH 7	pH 7	pH 7
120	pH 7	pH 7	pH 7	pH 8	pH 8
150	pH 7	pH 7	pH 7	pH 8	pH 8
180	pH 7	pH 7	pH 7	pH 9	pH 9
210	pH 7	pH 7	pH 7	pH 9	pH 9
240	pH 7	pH 7	pH 7	pH 9	pH 9
270	pH 7	pH 7	pH 8	pH 9	pH 9
300	pH 7	pH 7	pH 8	pH 9	pH 9

Table 1: Arginine test using pH indicator stick to measure pH changes of the media. Table represents the change in pH over the time span of 300 minutes i.e. 5 hours.

Here,

Control (1) - LB media
Control (2) - LB media with 10 mM of arginine
Control (3) - LB media plus B. subtilis 168
Test (1) - LB media with 10 mM of arginine plus B. subtilis 168
Test (2) - LB media with 10 mM of arginine plus B. subtilis 168

Conclusion

B. subtilis 168 breaks down arginine to urea by producing arginase. The urea is then further broken down to ammonia and carbonate ions by urease. This will lead to an increase in pH. Both the test 1 and test 2, which contain B. subtilis 168 and 10 mM of arginine show an increase in pH from 7 to 9. While the control 1 and control 2, which contain no B. subtilis 168 remains at pH 7. The control 3 which contain B. subtilis 168 but without addition of arginine show an increase in pH from 7 to 8. This could be due to unidentified products that are secreted by the bacteria.

Therefore this experiment indicates that B. subtilis 168 is able to utilise arginine, and thus increase the overall pH of the media. The production of carbonate ion will lead to its binding with calcium ions provided in the media leading to formation of calcium carbonate.

@@ Line 1: / Line 1: @@
-====Aims of the experiment====
+{{Team:Newcastle/mainbanner}}
-The aim of today's experiment is to extract genomic DNA from ''Bacillus subtilis'' strain 3610,genes from which will be needed for the swarming biobrick.
+=Arginine experiment=
-====Materials Required====
+==Aim==
+The aim of this experiment is to determine whether ''B. subtilis'' 168 is able to take up external arginine.
-*Cells grown from yesterday
+==Procedure==
-*Centrifuge
+* Please refer to [[Team:Newcastle/Arginine test| Arginine test]]
-*pipette
-*lysozyme
-*Cell lysis solution
-*RNAse solution
-*protein orecipitation solution
-*ice
-*isopropanol
-*ethanol
-====Procedure====
+==Results==
+Arginine is an amino acid that is positively charged. Therefore if ''B. subtilis'' 168 is able to take up arginine, it will cause a pH change in the media. This would result in an increase in pH.
-=====Cell lysis=====
+|[[Image:Newcastle_arginine_test_230710.png|500px ]]
-#Pellet cells by centrifugation at 3600rpm for 10 minutes
-#Pour off supernatant
-#Add 0.5ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5ml eppendorf tube
-#Add 25microlitres of lysozyme and invert 25 times
-#Incubate for 30minutes at 37°C inverting occasionally
-#Centrifuge at 13000rpm for 10minutes to pellet the cells then remove the supernatant
-#Add 0.5ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells
-#Heat sample for 30 mins mix every 5-10 mins
-=====RNase treatment=====
+'''Figure 1:''' Arginine test using pH indicator stick to measure pH changes of the media.
-#Add 3 microlitres of RNAse A solution to the cell lysate
+{|border=1
-#Mix by inverting 25 times and incubate at 37°C for 60 minutes
+|-
+!Time (in minutes)
+!Control (1)
+!Control (2)
+!Control (3)
+!Test (1)
+!Test (2)
+|-
+|0
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|-
+|30
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|-
+|60
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|-
+|90
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|pH 7
+|-
+|120
+|pH 7
+|pH 7
+|pH 7
+|pH 8
+|pH 8
+|-
+|150
+|pH 7
+|pH 7
+|pH 7
+|pH 8
+|pH 8
+|-
+|180
+|pH 7
+|pH 7
+|pH 7
+|pH 9
+|pH 9
+|-
+|210
+|pH 7
+|pH 7
+|pH 7
+|pH 9
+|pH 9
+|-
+|240
+|pH 7
+|pH 7
+|pH 7
+|pH 9
+|pH 9
+|-
+|270
+|pH 7
+|pH 7
+|pH 8
+|pH 9
+|pH 9
+|-
+|300
+|pH 7
+|pH 7
+|pH 8
+|pH 9
+|pH 9
+|}
+'''Table 1:''' Arginine test using pH indicator stick to measure pH changes of the media. Table represents the change in pH over the time span of 300 minutes i.e. 5 hours.
+Here,
+#Control (1) - LB media
+#Control (2) - LB media with 10 mM of arginine
+#Control (3) - LB media plus ''B. subtilis'' 168
+#Test (1) - LB media with 10 mM of arginine plus ''B. subtilis'' 168
+#Test (2) - LB media with 10 mM of arginine plus ''B. subtilis'' 168
+==Conclusion==
+''B. subtilis'' 168 breaks down arginine to urea by producing arginase. The urea is then further broken down to ammonia and carbonate ions by urease. This will lead to an increase in pH. Both the test 1 and test 2, which contain ''B. subtilis'' 168 and 10 mM of arginine show an increase in pH from 7 to 9. While the control 1 and control 2, which contain no ''B. subtilis'' 168 remains at pH 7. The control 3 which contain ''B. subtilis'' 168 but without addition of arginine show an increase in pH from 7 to 8. This could be due to unidentified products that are secreted by the bacteria.
+Therefore this experiment indicates that ''B. subtilis'' 168 is able to utilise arginine, and thus increase the overall pH of the media. The production of carbonate ion will lead to its binding with calcium ions provided in the media leading to formation of calcium carbonate.
+{{Team:Newcastle/footer}}

Team:Newcastle/23 July 2010

From 2010.igem.org

Latest revision as of 21:00, 27 October 2010

Contents

Arginine experiment

Aim

Procedure

Results

Conclusion