Team:Newcastle/7 September 2010

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=First transformation of ''B. subtilis'' 168 containing pMutin4 with pGFPrrnB containing ''yneA''=
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=Hyperspankoid characterisation=  
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===Aim===
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===Aims===
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The aim of the experiment is to perform insert the plasmid pGFP-rrnB containing ''yneA'' which have been ligated eariler into the chromosome of ''Bacillus subtilis'' 168. ''B. subtilis'' containing the intergated vector will be resistance to both the antibiotics chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain both the antibiotics. The second step will be to identify those colones that have the plasmid intergated at the corerct position in the chromosome, which is the amylase gene locus. Thus those that have intergrated at the wrong position will not be able to break down starch, which can be tested with the iodine test.
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The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of ''rfp'' on  the pSB1C3 plasmid seperately. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR.
===Materials and Protocol===
===Materials and Protocol===
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Please refer to: [[Team:Newcastle/Transformation of B. subtilis| Transformation of ''B. subtilis'']]
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Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] for Phusion PCR protocol.  
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Note: Overnight culture of ''B. subtilis'' 168 in MM competence medium was done the day before and the iodine test was performed the day after.
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===Result===
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The details of the four PCR reactions are included in the table below:
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The transformation was unsuccessful.
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=Subtilin Immunity BioBrick=
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=== Aims ===
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The aim of this experiment is to perform minipreps of the 16 overnight cultures that were left overnight from [[Team:Newcastle/6 September 2010#Subtilin_Immunity_BioBrick| yesterday]] for the Subtilin Immunity BioBrick.
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=== Methods ===
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The [[Team:Newcastle/Qiagen_Minipreps| Qiagen Miniprep]] protocol was used for each of the 16 cultures, followed by the 
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[[TeamNewcastleNanoDrop_Spectrophotometer| NanoDrop Spectrophotometer]] protocol.
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=== Results, Discussion and Conclusion ===
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The nanodrop spectrophotometer results for the 16 minipreps for Subtilin Immunity are below (units: ng/ml):
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{|border=1
{|border=1
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!'''Tube'''
!'''Tube'''
!'''Part to be amplified'''
!'''Part to be amplified'''
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!'''DNA fragment consisting the part'''
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!'''DNA fragment consisting the part i.e. template'''
!'''Forward primer'''
!'''Forward primer'''
!'''Reverse Primer'''
!'''Reverse Primer'''
!'''Melting Temperature (Tm in °C) '''
!'''Melting Temperature (Tm in °C) '''
!'''Size of the fragment (in bp)'''
!'''Size of the fragment (in bp)'''
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!'''Extension time* (in seconds)'''
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!'''Extension time (in seconds)'''
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|1
|1
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|Plasmid Vector
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|Hyperspankoid 
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|PSB1C3
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|yneA
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|P1V1 forward
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|Prom_for
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|P2V1 reverse
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|HpSpanPspacoid_rev
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|53.3
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|65
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|2046 +
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|150
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|70
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|15
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|-
|2
|2
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|Promoter and RBS (pVeg-SpoVG)
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|Pspacoid
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|BioBrick Bba_K143053
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|kinA
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|P1P1 forward
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|Prom_for
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|P2P2 reverse
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|HpSpanPspacoid_rev
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|51.7
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|65
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|139 +
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|150
|15
|15
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|3
|3
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|''spaIFEG'' Gene Cluster
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|Hyperspank
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|''B. subtilis'' ATCC 6633
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|K143055 from Bacillus plate - BS022
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|P1S1 forward
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|Prom_for
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|P2S1 reverse
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|Pspac-hy rev
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|46.0
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|62
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|2753 +
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|150
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|110
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|15
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|-
|4
|4
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|Double terminator
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|Plasmid
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|pSB1AK3 consisting BBa_B0014
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|vector pSB1C3/''rfp''
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|P1T1 forward
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|Vector/RFP_for
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|P2T1 reverse
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|P2-V1
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|50.9
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|64
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|116 +
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|2072
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|15
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|60
|}
|}
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#76.5
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'''Table 1''': The table shows the four different Phusion PCR reactions carried out for the characterisation of the hyperspankoid promoter.
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#61.5
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#89.9
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#87.6
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#6.3
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#69.5
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#81.3
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#70.2
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#73.3
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#63.0
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=Hyperspankoid characterisation=
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*PCR reactions of the four parts that were carried out [[Team:Newcastle/6 September 2010#Hyperspankoid characterisation| yesterday]] were carried out again today but with a control as well. Why?
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*Restriction digests of the four parts using ''Eco''R1.
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*Gel of the 5 amplified sequences with a control.
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==Materials and Protocol==
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Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]], [[Team:Newcastle/Restriction_digests| Restriction Digest]] and [[Team:Newcastle/Gel_electrophoresis| Gel Electrophoresis]] for Phusion PCR, Restriction Digest and Gel electrophoresis protocol.  
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===Results===
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GEL IMAGE
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===Results, Discussion and Conclusion===
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===Results, Discussion and Conclusion===  
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...
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Gel extraction will be carried out [[Team:Newcastle/8_September_2010#Hyperspankoid characterisation| tomorrow]].
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Please refer to:[[Team:Newcastle/7 September 2010#Hyperspankoid_characterisation| 8.09.2010]] for result, discussion and conclusion.
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'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 02:37, 28 October 2010

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Contents

Hyperspankoid characterisation

Aims

The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of rfp on the pSB1C3 plasmid seperately. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR.

Materials and Protocol

Please refer to Phusion PCR for Phusion PCR protocol.

The details of the four PCR reactions are included in the table below:

Tube Part to be amplified DNA fragment consisting the part i.e. template Forward primer Reverse Primer Melting Temperature (Tm in °C) Size of the fragment (in bp) Extension time (in seconds)
1 Hyperspankoid yneA Prom_for HpSpanPspacoid_rev 65 150 15
2 Pspacoid kinA Prom_for HpSpanPspacoid_rev 65 150 15
3 Hyperspank K143055 from Bacillus plate - BS022 Prom_for Pspac-hy rev 62 150 15
4 Plasmid vector pSB1C3/rfp Vector/RFP_for P2-V1 64 2072 60

Table 1: The table shows the four different Phusion PCR reactions carried out for the characterisation of the hyperspankoid promoter.

Results, Discussion and Conclusion

Please refer to: 8.09.2010 for result, discussion and conclusion.


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