Team:TU Delft/12 July 2010 content

From 2010.igem.org

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===Lab Work===
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==Lab work==
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'''Characterization of Anderson RBS sequences'''
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<h4>Characterization of Anderson RBS sequences</h4>
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''E.coli'' Top10 strains containing the following composite biobricks in pSB1A2 [https://2010.igem.org/Team:TU_Delft#/blog?blog=8_July_2010 had been obtained]:
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E.coli TOP 10 strains containing the following composite biobricks in pSB1A2 [https://2010.igem.org/Team:TU_Delft#/blog?blog=8_July_2010 had been obtained]:
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|<b> BioBrick # </b>
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|'''BioBrick'''
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|<b>Composed of: </b>
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|'''Composed of:'''
|-
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|[http://partsregistry.org/Part:BBa_K398500 K398500]
|[http://partsregistry.org/Part:BBa_K398500 K398500]
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|}
|}
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To prepare for the fluorescence assay measurements the strains were grown in M9 minimal medium containing 0.4% glucose and ampicillin.  
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To prepare for the fluorescence assay measurements the strains were grown in M9 minimal medium containing 0.4% glucose and 100 μg/mL Ampicillin.  
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High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus the fluorescence assays will also be performed with the biobricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB-Amp, awaiting QIAGEN Midi kit plasmid isolation and plasmid swapping.
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High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus we are planning to also measure the fluorescence assays with the BioBricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB with 100 μg/mL Ampicillin, awaiting plasmid isolation and plasmid swapping.
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'''Emulsifier'''
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<h4>Emulsifier</h4>
The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3.
The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3.
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Digestion substrates:
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Digested were performed according to the [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion protocol]]:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|<b>Label</b>
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|'''#'''
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|<b>Content</b>
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|'''Digestion reaction'''
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|<b>Concentration (ng/ul)</b>
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|'''Needed fragment'''
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|-
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| D1
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|1
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| Plasmid [http://partsregistry.org/Part:pSB1T3 pSB1T3]
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|1,0 μg AlnA + EcoRI + SpeI
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| 137.2
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|‘E – I13401 – S’
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|-
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| D2
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|2
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| AlnA
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|1,0 μg OprG + EcoRI + SpeI
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| 357.4
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|‘E – J61100 – S’
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|-
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| D3
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|3
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| OprG
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|1,0 μg R0011 + EcoRI + SpeI
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| 197.3
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|‘E – J61107 – S’
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|-
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| D4
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|4
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| B0015
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|1,0 μg B0032 + XbaI + PstI
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| 360.5
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|‘X – J61117 – P’
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|-
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| D5
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|5
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| R0011
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|1,0 μg B0015 + XbaI + PstI
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| 42.0
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|‘X – J61101 – P’
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|-
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| D6
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|6
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| B0032
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|1,0 μg pSB1T3 + EcoRI + PstI
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| 82.0
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|‘E – ---- – P’
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|}
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|}
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Digested according to the digestion protocol (Link needed!).
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The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] overnight to produce the following biobricks:
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The digestion products were ligated over night to produce the following biobricks:
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|<b>Label</b>
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|<b>BioBrick # </b>
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|<b>Composed of: </b>
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|-
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|L1
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|Control
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|Plasmid
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|-
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|L2
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|[http://partsregistry.org/Part:BBa_K398202 K398202]
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|R0011 + B0032
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|-
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|L3
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|[http://partsregistry.org/Part:BBa_K398203 K398203]
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|OprG + B0015
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|-
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|L4
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|[http://partsregistry.org/Part:BBa_K398204 K398204]
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|AlnA + B0015
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|}
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Revision as of 19:05, 17 July 2010

Lab work

Characterization of Anderson RBS sequences

E.coli Top10 strains containing the following composite biobricks in pSB1A2 had been obtained:

BioBrick Composed of:
K398500 J23100 + J61100 + I13401
K398501 J23100 + J61101 + I13401
K398502 J23100 + J61107 + I13401
K398503 J23100 + J61117 + I13401
K398504 J23100 + J61127 + I13401

To prepare for the fluorescence assay measurements the strains were grown in M9 minimal medium containing 0.4% glucose and 100 μg/mL Ampicillin.

High copy number RBS characterizations are rarely indicative of RiPS in system operation plasmids, thus we are planning to also measure the fluorescence assays with the BioBricks on pSB3C5. In order to obtain sufficient BioBrick DNA the strains were grown in 250 mL LB with 100 μg/mL Ampicillin, awaiting plasmid isolation and plasmid swapping.

Emulsifier

The bricks for emulsifier production were assembled. The stock plasmids containing AlnA, OprG, R0011 and B0032 were digested and ligated into plasmid pSB1T3.

Digested were performed according to the digestion protocol:

# Digestion reaction Needed fragment
1 1,0 μg AlnA + EcoRI + SpeI ‘E – I13401 – S’
2 1,0 μg OprG + EcoRI + SpeI ‘E – J61100 – S’
3 1,0 μg R0011 + EcoRI + SpeI ‘E – J61107 – S’
4 1,0 μg B0032 + XbaI + PstI ‘X – J61117 – P’
5 1,0 μg B0015 + XbaI + PstI ‘X – J61101 – P’
6 1,0 μg pSB1T3 + EcoRI + PstI ‘E – ---- – P’

The digestion products were ligated overnight to produce the following biobricks:

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