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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==Characterization of Anderson RBS sequences==
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''E.coli'' Top10 strains containing the following composite BioBricks in pSB1A2 [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=8_July_2010 had been obtained]:
-
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]:
+
{| 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_K398050 K398050]
+
|[http://partsregistry.org/Part:BBa_K398500 K398500]
|J23100 + J61100 + I13401
|J23100 + J61100 + I13401
|-
|-
-
|[http://partsregistry.org/Part:BBa_K398051 K398051]
+
|[http://partsregistry.org/Part:BBa_K398501 K398501]
|J23100 + J61101 + I13401
|J23100 + J61101 + I13401
|-
|-
-
|[http://partsregistry.org/Part:BBa_K398052 K398052]
+
|[http://partsregistry.org/Part:BBa_K398502 K398502]
|J23100 + J61107 + I13401
|J23100 + J61107 + I13401
|-  
|-  
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|[http://partsregistry.org/Part:BBa_K398053 K398053]
+
|[http://partsregistry.org/Part:BBa_K398503 K398503]
|J23100 + J61117 + I13401
|J23100 + J61117 + I13401
|-
|-
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|[http://partsregistry.org/Part:BBa_K398054 K398054]
+
|[http://partsregistry.org/Part:BBa_K398504 K398504]
|J23100 + J61127 + I13401  
|J23100 + J61127 + I13401  
|}
|}
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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.  
+
To prepare for the fluorescence assay measurements the strains were grown in 3 mL LB medium containing 100 μg/mL AMP as well as in M9 minimal medium containing 0.4% glucose and 100 μg/mL AMP.
-
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.
+
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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==Emulsifier==
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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|'''Sample'''
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|<b>Concentration (ng/ul)</b>
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|'''Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
 +
|'''Needed fragment'''
|-
|-
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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
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| 137.2
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|EcoRI
 +
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–AlnA–S’
|-
|-
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| D2
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|2
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| AlnA
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|1.0 μg OprG
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| 357.4
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|EcoRI
 +
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–OprG–S’
|-
|-
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| D3
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|3
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| OprG
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|1.0 μg R0011
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| 197.3
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|EcoRI
 +
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–R0011–S’
|-
|-
-
| D4
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|4
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| B0015
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|1.0 μg B0032
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| 360.5
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|XbaI
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–B0032–P’
|-
|-
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| D5
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|5
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| R0011
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|1.0 μg B0015
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| 42.0
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|XbaI
 +
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–B0015–P’
|-
|-
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| D6
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|6
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| B0032
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|1.0 μg pSB1T3
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| 82.0
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|EcoRI
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|}
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|PstI
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|2 (BioLabs)
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Digested according to the digestion protocol (Link needed!).
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|✓
 +
|‘E–linear pSB1T3–P’
 +
|}
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The digestion products were ligated over night to produce the following biobricks:
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The digestion products were [[Team:TU_Delft/protocols/ligation|ligated]] overnight:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
-
|<b>Label</b>
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|'''#'''
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|<b>BioBrick # </b>
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|'''BioBrick'''
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|<b>Composed of: </b>
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|'''Fragment 1'''
 +
|'''Fragment 2'''
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|'''Recipient vector'''
|-
|-
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|L1
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|1
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|Control
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|K398202
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|Plasmid
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|μL ‘E–R0011–S’
 +
|μL ‘X–B0032–P’
 +
|μL ‘E–linear pSB1T3–P’
|-
|-
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|L2
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|2
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|[http://partsregistry.org/Part:BBa_K398202 K398202]
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|K398203
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|R0011 + B0032
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|μL ‘E–AlnA–S’
 +
|μL ‘X–B0015–P’
 +
|μL ‘E–linear pSB1T3–P’
|-
|-
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|L3
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|3
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|[http://partsregistry.org/Part:BBa_K398203 K398203]
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|K398204
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|OprG + B0015
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|μL ‘E–OprG–S’
 +
|μL ‘X–B0015–P’
 +
|μL ‘E–linear pSB1T3–P’
|-
|-
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|L4
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|4
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|[http://partsregistry.org/Part:BBa_K398204 K398204]
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|negative control
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|AlnA + B0015
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|'''-'''
 +
|'''-'''
 +
|μL ‘E–linear pSB1T3–P’
|}
|}

Latest revision as of 12:38, 3 August 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:
[http://partsregistry.org/Part:BBa_K398500 K398500] J23100 + J61100 + I13401
[http://partsregistry.org/Part:BBa_K398501 K398501] J23100 + J61101 + I13401
[http://partsregistry.org/Part:BBa_K398502 K398502] J23100 + J61107 + I13401
[http://partsregistry.org/Part:BBa_K398503 K398503] J23100 + J61117 + I13401
[http://partsregistry.org/Part:BBa_K398504 K398504] J23100 + J61127 + I13401

To prepare for the fluorescence assay measurements the strains were grown in 3 mL LB medium containing 100 μg/mL AMP as well as in M9 minimal medium containing 0.4% glucose and 100 μg/mL AMP.

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:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1.0 μg AlnA EcoRI SpeI 2 (BioLabs) ✓ ‘E–AlnA–S’
2 1.0 μg OprG EcoRI SpeI 2 (BioLabs) ✓ ‘E–OprG–S’
3 1.0 μg R0011 EcoRI SpeI 2 (BioLabs) ✓ ‘E–R0011–S’
4 1.0 μg B0032 XbaI PstI 2 (BioLabs) ✓ ‘X–B0032–P’
5 1.0 μg B0015 XbaI PstI 2 (BioLabs) ✓ ‘X–B0015–P’
6 1.0 μg pSB1T3 EcoRI PstI 2 (BioLabs) ✓ ‘E–linear pSB1T3–P’

The digestion products were ligated overnight:

# BioBrick Fragment 1 Fragment 2 Recipient vector
1 K398202 μL ‘E–R0011–S’ μL ‘X–B0032–P’ μL ‘E–linear pSB1T3–P’
2 K398203 μL ‘E–AlnA–S’ μL ‘X–B0015–P’ μL ‘E–linear pSB1T3–P’
3 K398204 μL ‘E–OprG–S’ μL ‘X–B0015–P’ μL ‘E–linear pSB1T3–P’
4 negative control - - μL ‘E–linear pSB1T3–P’
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