Team:Newcastle/10 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Gel extraction of ''rocF'' BioBrick components=
 
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==Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3==
 
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===Aim===
 
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The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
 
-
===Materials and Protocol===
 
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
 
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===Result===
+
=Preparation of subtilin immunity BioBrick primers and Phusion PCR =
-
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution.
 
-
We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment. 
 
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{|border=1
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==Aims==
-
|-
+
-
!
+
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!'''Pspac_oid pormoter'''
+
-
!'''Double Terminator'''
+
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!'''Plasmid Vector pSB1C3'''
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-
|-
+
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|'''Size of the Fragment (in bp)'''
+
-
|148 approx.
+
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|116 approx.
+
-
|2072 approx.
+
-
|}
+
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'''Table 1''': Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
+
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===Discussion===
+
[[Image:Newcastle_6_primer_tubes.JPG|200px|right]]
-
We found bands  of appropriate sizes in their respective lanes.
+
To rehydrate the subtilin immunity primers. The six primers ordered are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already have in stock, therefore no rehydration will be needed. (Primer 1 = Forward primer and Primer 2 = Reverse primer).  
-
===Conclusion===
+
Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, ''spaIFEG'' gene cluster and double terminator.
-
Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
+
-
==Gel extraction of all 6 fragments for ''rocF''==
+
For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
-
==Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3==
+
==Materials and Protocol==
-
===Aim===
+
Please refer to the [[Team:Newcastle/DNA_re-hydration| DNA Re-hydration]] protocol.
-
The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
+
-
===Materials and Protocol===
+
Please refer to the [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] protocol.
-
Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
+
-
 
+
-
===Result===
+
-
 
+
-
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution.
+
-
We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment. 
+
{|border=1
{|border=1
|-
|-
-
!
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!'''Tube'''
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!'''Pspac_oid pormoter'''
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!'''Part to be amplified'''
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!'''Double Terminator'''
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!'''DNA fragment consisting the part'''
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!'''Plasmid Vector pSB1C3'''
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!'''Forward primer'''
 +
!'''Reverse Primer'''
 +
!'''Melting Temperature (Tm in °C) '''
 +
!'''Size of the fragment (in bp)'''
 +
!'''Extension time* (in seconds)'''
|-
|-
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|'''Size of the Fragment (in bp)'''
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|1
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|148 approx.
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|Plasmid Vector
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|116 approx.
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|PSB1C3
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|2072 approx.
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|P1V1 forward
 +
|P2V1 reverse
 +
|53.3
 +
|2046 +
 +
|70
 +
|-
 +
|2
 +
|Promoter and RBS (pVeg-SpoVG)
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|BioBrick Bba_K143053
 +
|P1P1 forward
 +
|P2P2 reverse
 +
|51.7
 +
|139 +
 +
|15
 +
|-
 +
|3
 +
|''spaIFEG'' Gene Cluster
 +
|''B. subtilis'' ATCC 6633
 +
|P1S1 forward
 +
|P2S1 reverse
 +
|46.0
 +
|2753 +
 +
|110
 +
|-
 +
|4
 +
|Double terminator
 +
|pSB1AK3 consisting BBa_B0014
 +
|P1T1 forward
 +
|P2T1 reverse
 +
|50.9
 +
|116 +
 +
|15
|}
|}
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'''Table 1''': Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
 
-
===Discussion===
+
'''Table 5''': This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
-
We found bands  of appropriate sizes in their respective lanes.
+
* The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
 +
* Note: The stock primer have to be handled with extra caution, because they will be the main stocks from which working stock solutions will be made from. Therefore, gloves have to be worn to prevent any contamination. Water will have to be from the Pure Lab Distilled Water.
-
===Conclusion===
+
==Discussion==
-
Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
+
-
==Gibson assembly of ''rocF'' BioBrick==
+
Gel electrophoresis for the PCR product will be done on[[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].
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=lacI and pVeg=
 
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==Aims==
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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We aim to [[Team:Newcastle/Qiagen_Minipreps| extract]] pVeg and lacI from transformed ''E.coli'' DH5alpha which was cultured in LB broth overnight(see [[Team:Newcastle/9_August_2010|09.08.2010]])check the concentration of DNA using [[TeamNewcastleNanoDrop_Spectrophotometer| nanodrop]] and perform a [[Team:Newcastle/Restriction_digests|restriction digest]] so we can run the samples on a [[Team:Newcastle/Gel_electrophoresis| gel]] to check the insert sizes.
+
-
 
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==Materials and Protocols==
+
-
 
+
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*[[Team:Newcastle/Qiagen_Minipreps|Plasmid extraction protocol]]
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*[[TeamNewcastleNanoDrop_Spectrophotometer|Nanodrop protocol]]
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*[[Team:Newcastle/Restriction_digests|Restriction digest protocol]]
+
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*[[Team:Newcastle/Gel_electrophoresis|Gel electrophoresis protocol]]
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*[[Team:Newcastle/Gel_extraction| Gel extraction protocol]]
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-
 
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See protocols for materials
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-
 
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==Results==
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-
 
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Results from Nanodrop:
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{|border=1
+
-
|-
+
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!
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!lacI 1
+
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!lacI 2
+
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!lacI 3
+
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!lacI 4
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!pVeg
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|-
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!Concentration of DNA ng/microlitre
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!122.5
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!139.4
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!130.0
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!150.0
+
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!155.0
+
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|}
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-
 
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=Subtilin Immunity BioBrick=
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-
 
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==Aims==
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-
 
+
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Subtilin is required as a cell-signalling molecule in order to trigger calcium carbonate precipitation, as well as bacteria filament formation. We aim to produce 2 subtilin BioBricks; Production and Immunity. Because subtilin is a lantibiotic that is found naturally in ''Bacillus subtilis'' ATCC 6633, the introduction of subtilin into our ''Bacillus'' 168 cells would actually kill them. Therefore, it is essential to establish immunity for 168 strains.
+
-
 
+
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==Materials and Protocol==
+
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Please refer to the [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] protocol.
+
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 23:06, 27 October 2010

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Contents

Preparation of subtilin immunity BioBrick primers and Phusion PCR

Aims

Newcastle 6 primer tubes.JPG

To rehydrate the subtilin immunity primers. The six primers ordered are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already have in stock, therefore no rehydration will be needed. (Primer 1 = Forward primer and Primer 2 = Reverse primer).

Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, spaIFEG gene cluster and double terminator.

For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)

Materials and Protocol

Please refer to the DNA Re-hydration protocol.

Please refer to the Phusion PCR protocol.

Tube Part to be amplified DNA fragment consisting the part Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time* (in seconds)
1 Plasmid Vector PSB1C3 P1V1 forward P2V1 reverse 53.3 2046 + 70
2 Promoter and RBS (pVeg-SpoVG) BioBrick Bba_K143053 P1P1 forward P2P2 reverse 51.7 139 + 15
3 spaIFEG Gene Cluster B. subtilis ATCC 6633 P1S1 forward P2S1 reverse 46.0 2753 + 110
4 Double terminator pSB1AK3 consisting BBa_B0014 P1T1 forward P2T1 reverse 50.9 116 + 15

Table 5: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.

  • The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
  • Note: The stock primer have to be handled with extra caution, because they will be the main stocks from which working stock solutions will be made from. Therefore, gloves have to be worn to prevent any contamination. Water will have to be from the Pure Lab Distilled Water.

Discussion

Gel electrophoresis for the PCR product will be done on 11 August.


Go back to our main Lab book page

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