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.

|

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.
-===Materials Required===
+=Arginine experiment=
-* Cells grown from yesterday
-* Centrifuge
-* pipette
-* lysozyme
-* Cell lysis solution
-* RNase solution
-* protein orecipitation solution
-* ice
-* isopropanol
-* ethanol
-===Procedure===
+==Aim==
+The aim of this experiment is to determine whether ''B. subtilis'' 168 is able to take up external arginine.
-=====Cell lysis=====
+==Procedure==
-# Pellet cells by centrifugation at 3600rpm for 10 minutes
+* Please refer to [[Team:Newcastle/Arginine test| Arginine test]]
-# 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=====
+==Results==
-# Add 3 microlitres of RNAse A solution to the cell lysate
+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.
-# Mix by inverting 25 times and incubate at 37°C for 60 minutes
-=====Protein precipitation=====
-# Cool samples on ice.
+|[[Image:Newcastle_arginine_test_230710.png|500px ]]
-# Add 0.5 ml protein precipitation solution to each tube.
-# Vortex vigorously at high speed for 20 seconds to miux the protein precipitation solution uniformly with the cell lysate. Place samples on ice for 5 minutes.
-# Centrifuge at 1300 rpm for 30 seconds or until the precipitated proteins form a tight pellet.
-=====DNA precipitation=====
+'''Figure 1:''' Arginine test using pH indicator stick to measure pH changes of the media.
-# Pour the supernatant containing the DNA into a clean eppendorf tube. (The samples may be kept at -20°C overnight at this stage.)
-# Add 0.5ml isopropanol to each tube.
-# Mix by inverting gently for 50 times.
-# Centrifuge at 1300 rpm for 1 minute. The DNA should be visible as a small white pellet.
-# Pour off the supernatant and drain the tube on clean absorbent paper. Add 0.5 ml 70% ethanol and invert tube several times to wash the DNA.
-# Centrifuge at 1300 rpm for 1 minute. Carefully pour off the ethanol.
-# Drain the tubes on clean absorbent paper. Allow to air dry for 10-15 minutes.
-=====DNA hydration=====
+{|border=1
-# Add 100 µl DNA hydration solution to each tube.
+|-
-# Rehydrate DNA by incubating the sample for 1 hour at 65°C and overnight at room temperature. Tap the tube periodically to aid in dispersing the DNA.
+!Time (in minutes)
-# For storage, centrifuge briefly and store at -20°C.
+!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