Team:TU Delft/13 July 2010 content

From 2010.igem.org

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(→Lab work)
 
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=Lab work=
=Lab work=
-
<h4>Ordered DNA</h4>
+
==Ordered DNA==
We have now stock of the ordered DNA, to make a real BioBrick of this DNA we are going to ligated it into the iGEM plasmid backbone SB1C3. First we [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]] the plasmids:
We have now stock of the ordered DNA, to make a real BioBrick of this DNA we are going to ligated it into the iGEM plasmid backbone SB1C3. First we [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digested]] the plasmids:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
-
|'''Digestion reaction'''
+
|'''Sample'''
-
|'''Used Buffer'''
+
|'''Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
|'''Needed fragment'''
|'''Needed fragment'''
|-
|-
|1
|1
-
|1 μg alkB2+ EcoRI + PstI
+
|1 μg alkB2
-
|Buffer 3 (BioLabs)
+
|EcoRI  
-
|‘E – alkB2– P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–alkB2–P’
|-
|-
|2
|2
-
|1 μg rubA3+ SpeI + PstI
+
|1 μg rubA3
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – rubA3– P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–rubA3–P’
|-
|-
|3
|3
-
|1 μg ladA + SpeI + PstI
+
|1 μg ladA
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – ladA – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–ladA–P’
|-
|-
|4
|4
-
|1 μg ADH + SpeI + PstI
+
|1 μg ADH
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – ADH – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–ADH–P’
|-
|-
|5
|5
-
|1 μg AlnA + SpeI + PstI
+
|1 μg AlnA
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – AlnA – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–AlnA–P’
|-
|-
|6
|6
-
|1 μg OprG + SpeI + PstI  
+
|1 μg OprG
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – OprG – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–OprG–P’
|-
|-
|7
|7
-
|1 μg AlkS + SpeI + PstI  
+
|1 μg AlkS
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – AlkS – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–AlkS–P’
|-
|-
|8
|8
-
|1 μg PalkB + SpeI + PstI
+
|1 μg PalkB
-
|Buffer 3 (BioLabs)  
+
|EcoRI
-
|‘E – PalkB – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–PalkB–P’
|-
|-
|9
|9
-
|1 μg PalkS12+ SpeI + PstI
+
|1 μg PalkS12
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – PalkS12– P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–PalkS12-P’
|-
|-
|10
|10
-
|1 μg PhPFDα + SpeI + PstI
+
|1 μg PhPFDα
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – PhPFDα – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–PhPFDα–P’
|-
|-
|11
|11
-
|1 μg PhPFDβ + SpeI + PstI
+
|1 μg PhPFDβ
-
|Buffer 3 (BioLabs)
+
|EcoRI
-
|‘E – PhPFDβ – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–PhPFDβ–P’
|-
|-
|12
|12
-
|3 μg pSB1C3 + EcoRI + PstI
+
|3 μg pSB1C3
-
|Buffer 3 (BioLabs)
+
|EcoRI  
-
|‘E – ---- – P’
+
|PstI
 +
|3 (BioLabs)
 +
|✗
 +
|‘E–linear pSB1C3–P’
|}
|}
-
<h4>Solvent Tolerance and Hydrocarbon Sensing</h4>
+
==Solvent Tolerance and Hydrocarbon Sensing==
Some BioBricks are in production. [[Team:TU_Delft/protocols/restriction_enzyme_digestion|Digestion reaction]]
Some BioBricks are in production. [[Team:TU_Delft/protocols/restriction_enzyme_digestion|Digestion reaction]]
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
-
|'''Digestion reaction'''
+
|'''Sample'''
-
|'''Used Buffer'''
+
|'''Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
|'''Needed fragment'''
|'''Needed fragment'''
|-
|-
|1
|1
-
|1 μg PhPFDα + EcoRI + SpeI
+
|1 μg PhPFDα
-
|Buffer 2 + BSA (BioLabs)
+
|EcoRI  
-
|‘E – PhPFDα – S’
+
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–PhPFDα–S’
|-
|-
|2
|2
-
|1 μg PhPFDβ + EcoRI + SpeI
+
|1 μg PhPFDβ
-
|Buffer 2 + BSA (BioLabs)
+
|EcoRI  
-
|‘E – PhPFDβ – S’
+
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–PhPFDβ–S’
|-
|-
|3
|3
-
|1 μg AlkS + EcoRI + SpeI
+
|1 μg AlkS  
-
|Buffer 2 + BSA (BioLabs)
+
|EcoRI  
-
|‘E – AlkS – S’
+
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–AlkS–S’
|-
|-
|4
|4
-
|1 μg PalkB + EcoRI + SpeI
+
|1 μg PalkB
-
|Buffer 2 + BSA (BioLabs)
+
|EcoRI  
-
|‘E – PalkB – S’
+
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E–PalkB–S’
|-
|-
|5
|5
-
|1 μg B0032 + XbaI + PstI
+
|1 μg B0032  
-
|Buffer 2 + BSA (BioLabs)
+
|XbaI
-
|‘X – B0032 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–B0032–P’
|-
|-
|6
|6
-
|1 μg B0015 + XbaI + PstI
+
|1 μg B0015  
-
|Buffer 2 + BSA (BioLabs)
+
|XbaI
-
|‘X – B0015 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–B0015–P’
|-
|-
|7
|7
-
|1 μg E0422 + XbaI + PstI
+
|1 μg E0422
-
|Buffer 2 + BSA (BioLabs)  
+
|XbaI
-
|‘X – E0422 – P’
+
|PstI
 +
|2 (BioLabs)
 +
|✓
 +
|‘X–E0422–P’
|-
|-
|8
|8
-
|3 μg pSB1T3 + EcoRI + PstI
+
|3 μg pSB1T3
-
|Buffer 2 + BSA (BioLabs)
+
|EcoRI  
-
|‘E - ---- - P’
+
|SpeI
 +
|2 (BioLabs)
 +
|✓
 +
|‘E-linear pSB1T3-P’
|}
|}
-
<h4>Emulsifier</h4>
+
==Emulsifier==
Today the digestion products from [[Team:TU_Delft/12_July_2010|yesterday]] were run on gel to see whether the plasmids were cut in the right way.
Today the digestion products from [[Team:TU_Delft/12_July_2010|yesterday]] were run on gel to see whether the plasmids were cut in the right way.
-
[[Image:TU_Delft_2010-07-13_P_Digestion.png|thumb|left|600px|1% Agarose gel runned at 100V for 1h]]
+
[[Image:TU_Delft_2010-07-13_P_Digestion.png|thumb|left|600px|1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
-
[[Image:TU_Delft_SmartLadder.jpg|thumb|right|EuroGentec SmartLadder]]
+
Lane description:
-
 
+
-
Lane description
+
-
:
+
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
|'''Description'''
|'''Description'''
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
 +
|'''Status'''
 +
|'''Remarks'''
|-
|-
|1
|1
-
|Biorad EZ marker (5 μL)
+
|Biorad EZ marker  
 +
|n/a
 +
|n/a
|
|
|-
|-
|2
|2
-
|Undigested pSB1T3 (10 μL + 2 μL loadingbuffer)
+
|Undigested pSB1T3  
 +
|3507
 +
|<font color=limegreen>✓</font>
|
|
|-
|-
|3
|3
-
|Digested pSB1T3 (10 μL + 2 μL loadingbuffer)
+
|pSB1T3 + EcoRI + PstI
-
|2500
+
|1085, 2422
 +
|<font color=limegreen>✓</font>
 +
|
|-
|-
|4
|4
-
|Undigested AlnA (10 μL + 2 μL loadingbuffer)
+
|Undigested AlnA  
 +
|3469
 +
|<font color=limegreen>✓</font>
|
|
|-
|-
|5
|5
-
|Digested AlnA (10 μL + 2 μL loadingbuffer)
+
|AlnA + EcoRI + SpeI
-
|1107
+
|1071, 2398
 +
|<font color=limegreen>✓</font>
 +
|
|-
|-
|6
|6
-
|Undigested OprG (10 μL + 2 μL loadingbuffer)
+
|Undigested OprG  
 +
|3122
 +
|<font color=limegreen>✓</font>
|
|
|-
|-
|7
|7
-
|Digested OprG (10 μL + 2 μL loadingbuffer)
+
|OprG + EcoRI + SpeI
-
|744
+
|708, 2414
 +
|<font color=limegreen>✓</font>
 +
|
|-
|-
|8
|8
-
|Undigested B0015 (10 μL + 2 μL loadingbuffer)
+
|Undigested B0015
 +
|3318
 +
|<font color=limegreen>✓</font>
|
|
|-
|-
|9
|9
-
|Digested B0015 (10 μL + 2 μL loadingbuffer)
+
|B0015 + XbaI + PstI
-
|130
+
|155, 3163
 +
|<font color=limegreen>✓</font>
 +
|fragment probably run of the gel
|-
|-
|10
|10
-
|Undigested R0011 (10 μL + 2 μL loadingbuffer) '''!'''
+
|Undigested R0011
-
|
+
|2134
 +
|<font color=limegreen>✓</font>
 +
|Sample not fully loaded on gel
|-
|-
|11
|11
-
|Digested R0011 (10 μL + 2 μL loadingbuffer)
+
|R0011 = EcoRI + SpeI
-
|55
+
|78, 2056
 +
|?
 +
|fragment probably run of the gel
|-
|-
|12
|12
-
|Undigested B0032 (10 μL + 2 μL loadingbuffer)
+
|Undigested B0032  
 +
|2092
 +
|<font color=limegreen>✓</font>
|
|
|-
|-
|13
|13
-
|Digested B0032 (10 μL + 2 μL loadingbuffer)
+
|B0032 + XbaI + PstI
-
|13
+
|39, 2053
 +
|?
 +
|fragment probably run of the gel
|-
|-
|14
|14
-
|SmartLadder (5 μL)
+
|SmartLadder  
 +
|n/a
 +
|n/a
|
|
|}
|}
-
 
-
'''!''' Sample not fully loaded on gel
 
-
 
-
The band position of the digestion products of AlnA and OprG correspond to their length. The other fragments (R0011, B0032 and B0015) probably run of the gel.
 
The ligation products that were incubated over night were transformed to Top10 competent cells according to the [[Team:TU_Delft/protocols/transformation|protocol]].
The ligation products that were incubated over night were transformed to Top10 competent cells according to the [[Team:TU_Delft/protocols/transformation|protocol]].
Line 199: Line 288:
==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
-
The first attempt at measuring fluorescence was made in adherence with the [http://partsregistry.org/Part:BBa_K176011|protocol] proposed by the USTC team of 2009:
+
The first attempt at measuring fluorescence was made in adherence with the [http://partsregistry.org/Part:BBa_K176011 protocol] proposed by the USTC team of 2009:
 +
 
 +
1. The overnight cultures of ''E.coli'' Top10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 (LB, 37 ℃, 160 rpm) were measured for OD600 (see table below) and diluted 1:100.
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''OD600'''
 +
|-
 +
|K398500
 +
|1.443
 +
|-
 +
|K398501
 +
|1.410
 +
|-
 +
|K398502
 +
|1.448
 +
|-
 +
|K398503
 +
|1.414
 +
|-
 +
|K398504
 +
|1.552
 +
|-
 +
|I13401
 +
|1.451
 +
|}
 +
 
 +
2. 100 μL and 300 μL aliquots of the diluted cultures were pipetted into 96-well plates in three-fold.
-
1. The overnight cultures of E.coli TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 (3 mL LB, 37℃, 200 rpm) were measured for OD600 and diluted 1:100.  
+
3. The plate was incubated at 37 ℃ while shaking for 3 hours.
-
2. 100 uL and 300 uL aliquots of the diluted cultures were pipetted into 96-well plates in three-fold.
+
4. The fluorescence and OD600 were measured for 16.30 hours with intervals 10 minutes by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm. LB medium was used as blank reference.
-
3. The fluorescence and OD600 were measured for 16.30 hours with intervals of 0.10 hours by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm.
+
Note: surprisingly no over night growth of the cultures was observed in M9 minimal medium containing glucose. This a larger volume of inoculate (in LB) was used for a second stab at over night growth in M9.
-
Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Midiprep kit from QIAGEN yielding sufficient plasmid DNA. These BioBricks can in future be placed under control of a low to medium copy number plasmid for comparison with high copy-number plasmid expression.
+
Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the [[Team:TU_Delft/protocols/midi-prep_plasmid_isolation|Qiagen Midi-prep plasmid isolation kit]] yielding sufficient plasmid DNA. These BioBricks can in future be placed under control of a low to medium copy number plasmid for comparison with high copy-number plasmid expression.

Latest revision as of 19:57, 5 August 2010

Contents

Lab work

Ordered DNA

We have now stock of the ordered DNA, to make a real BioBrick of this DNA we are going to ligated it into the iGEM plasmid backbone SB1C3. First we digested the plasmids:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1 μg alkB2 EcoRI PstI 3 (BioLabs) ✗ ‘E–alkB2–P’
2 1 μg rubA3 EcoRI PstI 3 (BioLabs) ✗ ‘E–rubA3–P’
3 1 μg ladA EcoRI PstI 3 (BioLabs) ✗ ‘E–ladA–P’
4 1 μg ADH EcoRI PstI 3 (BioLabs) ✗ ‘E–ADH–P’
5 1 μg AlnA EcoRI PstI 3 (BioLabs) ✗ ‘E–AlnA–P’
6 1 μg OprG EcoRI PstI 3 (BioLabs) ✗ ‘E–OprG–P’
7 1 μg AlkS EcoRI PstI 3 (BioLabs) ✗ ‘E–AlkS–P’
8 1 μg PalkB EcoRI PstI 3 (BioLabs) ✗ ‘E–PalkB–P’
9 1 μg PalkS12 EcoRI PstI 3 (BioLabs) ✗ ‘E–PalkS12-P’
10 1 μg PhPFDα EcoRI PstI 3 (BioLabs) ✗ ‘E–PhPFDα–P’
11 1 μg PhPFDβ EcoRI PstI 3 (BioLabs) ✗ ‘E–PhPFDβ–P’
12 3 μg pSB1C3 EcoRI PstI 3 (BioLabs) ✗ ‘E–linear pSB1C3–P’

Solvent Tolerance and Hydrocarbon Sensing

Some BioBricks are in production. Digestion reaction

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1 μg PhPFDα EcoRI SpeI 2 (BioLabs) ✓ ‘E–PhPFDα–S’
2 1 μg PhPFDβ EcoRI SpeI 2 (BioLabs) ✓ ‘E–PhPFDβ–S’
3 1 μg AlkS EcoRI SpeI 2 (BioLabs) ✓ ‘E–AlkS–S’
4 1 μg PalkB EcoRI SpeI 2 (BioLabs) ✓ ‘E–PalkB–S’
5 1 μg B0032 XbaI PstI 2 (BioLabs) ✓ ‘X–B0032–P’
6 1 μg B0015 XbaI PstI 2 (BioLabs) ✓ ‘X–B0015–P’
7 1 μg E0422 XbaI PstI 2 (BioLabs) ✓ ‘X–E0422–P’
8 3 μg pSB1T3 EcoRI SpeI 2 (BioLabs) ✓ ‘E-linear pSB1T3-P’

Emulsifier

Today the digestion products from yesterday were run on gel to see whether the plasmids were cut in the right way.

1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status Remarks
1 Biorad EZ marker n/a n/a
2 Undigested pSB1T3 3507 ✓
3 pSB1T3 + EcoRI + PstI 1085, 2422 ✓
4 Undigested AlnA 3469 ✓
5 AlnA + EcoRI + SpeI 1071, 2398 ✓
6 Undigested OprG 3122 ✓
7 OprG + EcoRI + SpeI 708, 2414 ✓
8 Undigested B0015 3318 ✓
9 B0015 + XbaI + PstI 155, 3163 ✓ fragment probably run of the gel
10 Undigested R0011 2134 ✓ Sample not fully loaded on gel
11 R0011 = EcoRI + SpeI 78, 2056 ? fragment probably run of the gel
12 Undigested B0032 2092 ✓
13 B0032 + XbaI + PstI 39, 2053 ? fragment probably run of the gel
14 SmartLadder n/a n/a

The ligation products that were incubated over night were transformed to Top10 competent cells according to the protocol.

Characterization of Anderson RBS sequences

The first attempt at measuring fluorescence was made in adherence with the [http://partsregistry.org/Part:BBa_K176011 protocol] proposed by the USTC team of 2009:

1. The overnight cultures of E.coli Top10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 (LB, 37 ℃, 160 rpm) were measured for OD600 (see table below) and diluted 1:100.

# OD600
K398500 1.443
K398501 1.410
K398502 1.448
K398503 1.414
K398504 1.552
I13401 1.451

2. 100 μL and 300 μL aliquots of the diluted cultures were pipetted into 96-well plates in three-fold.

3. The plate was incubated at 37 ℃ while shaking for 3 hours.

4. The fluorescence and OD600 were measured for 16.30 hours with intervals 10 minutes by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm. LB medium was used as blank reference.

Note: surprisingly no over night growth of the cultures was observed in M9 minimal medium containing glucose. This a larger volume of inoculate (in LB) was used for a second stab at over night growth in M9.

Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Qiagen Midi-prep plasmid isolation kit yielding sufficient plasmid DNA. These BioBricks can in future be placed under control of a low to medium copy number plasmid for comparison with high copy-number plasmid expression.

Retrieved from "http://2010.igem.org/Team:TU_Delft/13_July_2010_content"