==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.
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===Materials and Protocol===
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Please refer to: [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]].
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===Result===
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=Preparation of subtilin immunity BioBrick primers and Phusion PCR =
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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.
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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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|-
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!
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!'''Pspac_oid pormoter'''
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!'''Double Terminator'''
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!'''Plasmid Vector pSB1C3'''
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|-
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|'''Size of the Fragment (in bp)'''
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|148 approx.
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|116 approx.
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|2072 approx.
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|}
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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===
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We found bands of appropriate sizes in their respective lanes.
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===Conclusion===
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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.
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==Gel extraction of all 6 fragments for ''rocF''==
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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.
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===Materials and Protocol===
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Please refer to: [[Team:Newcastle/Gel extraction| Gel extraction]].
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===Result===
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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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|-
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!
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!'''Pspac_oid pormoter'''
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!'''Double Terminator'''
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!'''Plasmid Vector pSB1C3'''
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|-
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|'''Size of the Fragment (in bp)'''
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|148 approx.
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|116 approx.
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|2072 approx.
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|}
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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===
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We found bands of appropriate sizes in their respective lanes.
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===Conclusion===
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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.
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==Gibson assembly of ''rocF'' BioBrick==
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=lacI and pVeg=
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==Aims==
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We aim to [[Team:Newcastle/Qiagen_Minipreps| extract]] pVeg and lacI from transformed ''E.coli'' DH5α 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.
*[[Team:Newcastle/Gel_extraction| Gel extraction protocol]]
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See protocols for materials
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==Results==
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Results from Nanodrop:
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{|border=1
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|-
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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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=Subtilin Immunity BioBrick=
==Aims==
==Aims==
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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.
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).
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Lab work today will involve the rehydration of the primers that have arrived today. These primers 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.
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Phusion PCR will then be performed, using four different DNA fragments, the plasmid vector, promotor & RBS, ''spaIFEG'' gene cluster and double terminator.
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After the primers are ready, Phusion PCR will be performed, using four different template DNA: Plasmid Vector, Promotor & RBS, ''spaIFEG'' gene cluster and Double Terminator.
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For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
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Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water.
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==Materials and Protocol==
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Please refer to the [[Team:Newcastle/DNA_re-hydration| DNA Re-hydration]] protocol.
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==Materials and Protocol==
Please refer to the [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] protocol.
Please refer to the [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] protocol.
'''Table #''': Table # shows the four different Phusion PCR reactions that were carried out today. If this is succesful, the four parts can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
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'''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.
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* The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
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* The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
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* For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity.
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* 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.
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==Discussion==
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Gel electrophoresis for the PCR product will be done on[[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].
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==Results, Discussion and Conclusion==
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For results, discussion and conclusion, please refer to [[Team:Newcastle/11_August_2010#Subtilin_Immunity_BioBrick| 11 August]].
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
Preparation of subtilin immunity BioBrick primers and Phusion PCR
Aims
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)
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.