We started working on the Levans glue. For this we used agar plate containing 10% sucrose and ''Bacillus sphaericus'' strain was grown onto that medium. The sucrose cultures and controls were compared. The cultures grown on sucrose should be considerably thicker.
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Today we checked for transformants from the filamentous ''Bacillus subtilis'' transformation (with pMutin4) we performed yesterday.
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The overnight cultures of the different strains were also grown on solid agar plates with and without sucrose.
The aim of these experiments is to ligate the promoters hyperspankoid, pspacoid and hyperspank in front of RFP of the pSB1C3 plasmid. In order to do this, the first step is to amplify the three promoter sequences and the pSB1C3 plasmid using Phusion PCR and the different primers and template DNA.
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===Materials and Protocol===
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Please refer to [[Team:Newcastle/PCR#Phusion_PCR| Phusion PCR]] for Phusion PCR protocol.
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The details of the four PCR reactions are included in the table below:
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{|border=1
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|-
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!'''Tube'''
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!'''Part to be amplified'''
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!'''DNA fragment consisting the part i.e. template'''
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!'''Forward primer'''
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!'''Reverse Primer'''
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!'''Melting Temperature (Tm in °C) '''
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!'''Size of the fragment (in bp)'''
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!'''Extension time (in seconds)'''
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|-
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|1
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|Hyperspankoid
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|yneA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|
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|15
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|-
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|2
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|Pspacoid
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|kinA
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|Prom_for
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|HpSpanPspacoid_rev
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|65
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|
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|15
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|-
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|3
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|Hyperspank
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|K143055 from Bacillus plate - BS022
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|Prom_for
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|Pspac-hy rev
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|62
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|
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|15
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|-
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|4
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|Plasmid
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|vector pSB1C3/RFP
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|Vector/RFP_for
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|P2-V1
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|64
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|
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|60
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|}
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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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===Results, Discussion and Conclusion===
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...
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Please see [[Team:Newcastle/7 September 2010#Hyperspankoid_characterisation| tomorrow]]
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We checked our transformants under the light microscope, expecting to see a non-filamentous phenotype since the cells had not been induced with IPTG. The resutls were as expected.
Today we checked for transformants from the filamentous Bacillus subtilis transformation (with pMutin4) we performed yesterday.
We checked our transformants under the light microscope, expecting to see a non-filamentous phenotype since the cells had not been induced with IPTG. The resutls were as expected.
"
