Team:TU Delft/15 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Characterization of Anderson RBS sequences)
 
(21 intermediate revisions not shown)
Line 1: Line 1:
=Lab work=
=Lab work=
-
<h4>Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing</h4>
+
==Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing==
-
Some of transformations of [http://2010.igem.org/Team:TU_Delft#/blog?blog=14_July_2010 yesterday] containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a [[Team:TU_Delft/protocols/colony PCR|colony PCR]] overnight to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.
+
Some of transformations of [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=14_July_2010 yesterday] containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a [[Team:TU_Delft/protocols/colony PCR|colony PCR]] overnight to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.
-
===Emulsifier===
+
==Emulsifier==
-
The results from the Colony PCR were not conclusive [http://2010.igem.org/Team:TU_Delft#/blog?blog=14_July_2010 yesterday]. That is why we decided to isolate the plasmid using [[Team:TU_Delft/protocols/birnboim_plasmid_isolation|Birnboim protocol]]. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
+
The results from the Colony PCR were not conclusive [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=14_July_2010 yesterday]. That is why we decided to isolate the plasmid using [[Team:TU_Delft/protocols/birnboim_plasmid_isolation|Birnboim protocol]]. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
   
   
The following plasmid concentrations were obtained:  
The following plasmid concentrations were obtained:  
Line 14: Line 14:
|-
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398203 K398203]
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398203 K398203]
-
| OprG-B0015 (transformant 6)
+
| OprG-B0015 (6)
|
|
|-
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398203 K398203]
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398203 K398203]
-
| OprG-B0015 (transformant 7)
+
| OprG-B0015 (7)
|
|
|-
|-
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398204 K398204]
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398204 K398204]
-
| AlnA-B0015 (transformant 11)
+
| AlnA-B0015 (11)
|
|
|}
|}
Line 30: Line 30:
{| 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'''
|-
|-
|1
|1
|1.5 μg pSB1T3
|1.5 μg pSB1T3
-
|Buffer 2 + BSA (BioLabs)
+
|
 +
|
 +
|2 (BioLabs)  
 +
|✓
|-
|-
|2
|2
-
|1 μg OprG-B0015 (transformant 6)
+
|1 μg OprG-B0015 (6)
-
|Buffer 2 + BSA (BioLabs)
+
|
 +
|
 +
|2 (BioLabs)  
 +
|✓
|-
|-
|3
|3
-
|1 μg OprG-B0015 (transformant 7)
+
|1 μg OprG-B0015 (7)
-
|Buffer  2 + BSA (BioLabs)
+
|
 +
|
 +
|2 (BioLabs)  
 +
|✓
|-
|-
|4
|4
-
|1 μg AlnA-B0015 (transformant 11)
+
|1 μg AlnA-B0015 (11)
-
|Buffer  2 + BSA (BioLabs)
+
|
 +
|
 +
|2 (BioLabs)  
 +
|✓
|}
|}
[[Team:TU_Delft/protocols/agarose_gel |1% Agarose gel]] of digestion products:
[[Team:TU_Delft/protocols/agarose_gel |1% Agarose gel]] of digestion products:
-
[[Image:TU_Delft_2010-07-15_Digestion.png|300px|thumb|left|1% Agarose gel of digestion products]]
+
 
 +
[[Image:TU_Delft_2010-07-15_Digestion.png|300px|thumb|left|1 % agarose of plasmid check using digestion reaction. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded of digested plasmids, 5 μL sample + 1 μL loadingbuffer of undigested plasmids and 5 μL was loaded of marker]]
Lane description:
Lane description:
Line 58: Line 74:
|'''Description'''
|'''Description'''
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
 +
|'''Status'''
 +
|-
 +
|M1
 +
|SmarLadder marker
 +
|n/a
 +
|n/a
|-
|-
|1
|1
-
|SmarLadder marker (5 μL)
+
|Undigested pSB1T3
-
|
+
|3507
 +
|<font color=limegreen>✓</font>
|-
|-
|2
|2
-
|Undigested pSB1T3 (5 μL + 1 μL loadingbuffer)
+
|Digested pSB1T3  
-
|
+
|1972, 1176
 +
|<font color=red>✗</font>
|-
|-
|3
|3
-
|Digested pSB1T3 (10 μL + 1 μL loadingbuffer)
+
|Undigested OprG-B0015 (6)
-
|1972, 1176
+
|3285
 +
|<font color=limegreen>✓</font>
|-
|-
|4
|4
-
|Undigested OprG-B0015 (transformant 6) (5 μL + 1 μL loadingbuffer)
+
|Digested OprG-B0015 (6)  
-
|
+
|1828, 1475, 222
 +
|<font color=limegreen>✓</font>
|-
|-
|5
|5
-
|Digested OprG-B0015 (transformant 6) (10 μL + 2 μL loadingbuffer)
+
|Digested OprG-B0015 (7)
|1828, 1475, 222  
|1828, 1475, 222  
 +
|<font color=limegreen>✓</font>
|-
|-
|6
|6
-
|Digested OprG-B0015 (transformant 7) (10 μL + 2 μL loadingbuffer)
+
|Undigested AlnA-B0015 (11)  
-
|1828, 1475, 222
+
|3648
 +
|<font color=limegreen>✓</font>
|-
|-
|7
|7
-
|Undigested AlnA-B0015 (transformant 11) (5 μL + 1 μL loadingbuffer)
+
|Digested AlnA-B0015 (11)
-
|
+
|3511
-
|-
+
|<font color=limegreen>✓</font>
-
|8
+
-
|Digested AlnA-B0015 (transformant 11) (10 μL + 2 μL loadingbuffer)
+
-
|3511
+
|}
|}
==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
-
<h5>Fluorescence measurements attempt #2:</h5>
+
====Fluorescence measurements attempt #2:====
-
1. [http://2010.igem.org/Team:TU_Delft#/blog?blog=14_July_2010 Last night's] cultures of TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 were measured for biomass (OD600) and diluted to an OD600 of 0.1.  
+
1. [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=14_July_2010 Last night's] cultures of TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 were measured for biomass (OD600) and diluted to an OD600 of about 0.1 (see exact OD600 measurement of dilutions in table below).
-
2. Aliquots of 175 uL of the dilutions were pipetted into the wells of a 96-wells plate in three-fold.
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''OD600'''
 +
|-
 +
|K398500
 +
|0.102
 +
|-
 +
|K398501
 +
|0.108
 +
|-
 +
|K398502
 +
|0.108
 +
|-
 +
|K398503
 +
|0.107
 +
|-
 +
|K398504
 +
|0.098
 +
|-
 +
|I13401
 +
|0.109
 +
|}
 +
 
 +
2. Aliquots of 175 μL of the dilutions were pipetted into the wells of a 96-wells plate in three-fold. The plate was covered with a transparent film to prevent evaporation.
3. 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.
3. 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.
-
<h5>Assembly of reference construct:</h5>
+
'''Assembly of reference construct:'''
-
K081005 yielded no transformants and was thus transformed a second time.
+
K081005 yielded no transformants and was thus transformed a second time on ampicillin plates.
-
Transformants of J23100 were single colony PCRed (see gel image) and used as inoculate for 5 mL of LB for later plasmid isolation and [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
+
Transformants of J23100 were single colony PCRed (see gel image below) and used as inoculate for 5 mL of LB for later plasmid isolation and [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
-
The PCR amplification product of K081005 was digested using EcoRI and SpeI.  
+
The PCR amplification product of K081005 was digested using EcoRI and SpeI.
-
2 ug of I13401 in pSB1A2 was digested using EcoRI and XbaI.  
+
2 μg of I13401 in pSB1A2 was [[Team:TU_Delft/protocols/restriction enzyme digestion|digested]] using EcoRI and XbaI.
-
<h5>Positive control</h5>
+
====Positive control====
-
Transformants of I13522 were single colony PCRed (see gel image) and used as inoculate for 5 mL of LB for later plasmid isolation and [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
+
Transformants of I13522 were [[Team:TU_Delft/protocols/Colony_PCR|single colony PCRed]] (see gel image below) and used as inoculate for 5 mL of LB for later plasmid isolation and -80 °C stocks.
-
<h5>Gels of the day</h5>
 
[[Team:TU_Delft/protocols/agarose_gel |1% Agarose gel]] of scPCR product:
[[Team:TU_Delft/protocols/agarose_gel |1% Agarose gel]] of scPCR product:
-
[[Image:TU_Delft_2010_Colony_PCR_16_July.png|200px|thumb|left|1% Agarose gel of scPCR]]
+
[[Image:TU_Delft_2010_Colony_PCR_16_July.png|200px|thumb|left|1 % agarose of colony PCR. Gel runned 1 hour at 100 V.
 +
Of all samples 5 μL + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
-
Lane description:
+
Lane descriptions:
{|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)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|-
 +
|M1
 +
|SmartLadder marker
 +
|n/a
 +
|n/a
 +
|n/a
|-
|-
|1
|1
-
|SmarLadder marker (5 μL)
+
|transformant #1 of J23100
|
|
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|-
|-
|2
|2
-
|Undigested pSB1T3 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of J23100
|
|
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|-
|-
|3
|3
-
|Digested pSB1T3 (10 μL + 1 μL loadingbuffer)
+
|transformant #1 of I13522
-
|1972, 1176
+
-
|-
+
-
|4
+
-
|Undigested OprG-B0015 (transformant 6) (5 μL + 1 μL loadingbuffer)
+
|
|
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|-
|-
-
|5
+
|M2
-
|Digested OprG-B0015 (transformant 6) (10 μL + 2 μL loadingbuffer)
+
|EZ Load Ladder (5 μL)
-
|1828, 1475, 222
+
|n/a
-
|-
+
|n/a
-
|6
+
|n/a
-
|Digested OprG-B0015 (transformant 7) (10 μL + 2 μL loadingbuffer)
+
-
|1828, 1475, 222
+
-
|-
+
-
|7
+
-
|Undigested AlnA-B0015 (transformant 11) (5 μL + 1 μL loadingbuffer)
+
-
|
+
-
|-
+
-
|8
+
-
|Digested AlnA-B0015 (transformant 11) (10 μL + 2 μL loadingbuffer)
+
-
|3511
+
|}
|}
 +
 +
 +
From the gel it can be concluded that the transformants carry the desired plasmids with the proper BioBrink inserts.

Latest revision as of 20:02, 5 August 2010

Contents

Lab work

Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing

Some of transformations of yesterday containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a colony PCR overnight to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.

Emulsifier

The results from the Colony PCR were not conclusive yesterday. That is why we decided to isolate the plasmid using Birnboim protocol. We used 3 mL of the bacterial cells to make -80 °C stocks.

The following plasmid concentrations were obtained:

BioBrick Composed of Concentration (ng/μL)
K398203 OprG-B0015 (6)
K398203 OprG-B0015 (7)
K398204 AlnA-B0015 (11)

The plasmids were subsequently digested with an enzyme that characteristic digest for all different plasmids. The plasmids were digested with HindIII. This should yield 3 bands in the lane with the plasmid pSB1T3 containing J04450 (neg control), 1 band in the lane with AlnA-B0015 and 2 bands in the lane with OprG-B0015 on gel.

# Sample Enzyme 1 Enzyme 2 Buffer BSA
1 1.5 μg pSB1T3 2 (BioLabs)
2 1 μg OprG-B0015 (6) 2 (BioLabs)
3 1 μg OprG-B0015 (7) 2 (BioLabs)
4 1 μg AlnA-B0015 (11) 2 (BioLabs)

1% Agarose gel of digestion products:

1 % agarose of plasmid check using digestion reaction. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded of digested plasmids, 5 μL sample + 1 μL loadingbuffer of undigested plasmids and 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status
M1 SmarLadder marker n/a n/a
1 Undigested pSB1T3 3507
2 Digested pSB1T3 1972, 1176
3 Undigested OprG-B0015 (6) 3285
4 Digested OprG-B0015 (6) 1828, 1475, 222
5 Digested OprG-B0015 (7) 1828, 1475, 222
6 Undigested AlnA-B0015 (11) 3648
7 Digested AlnA-B0015 (11) 3511

Characterization of Anderson RBS sequences

Fluorescence measurements attempt #2:

1. Last night's cultures of TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 were measured for biomass (OD600) and diluted to an OD600 of about 0.1 (see exact OD600 measurement of dilutions in table below).

# OD600
K398500 0.102
K398501 0.108
K398502 0.108
K398503 0.107
K398504 0.098
I13401 0.109

2. Aliquots of 175 μL of the dilutions were pipetted into the wells of a 96-wells plate in three-fold. The plate was covered with a transparent film to prevent evaporation.

3. 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.

Assembly of reference construct: K081005 yielded no transformants and was thus transformed a second time on ampicillin plates. Transformants of J23100 were single colony PCRed (see gel image below) and used as inoculate for 5 mL of LB for later plasmid isolation and -80 °C stocks.

The PCR amplification product of K081005 was digested using EcoRI and SpeI. 2 μg of I13401 in pSB1A2 was digested using EcoRI and XbaI.

Positive control

Transformants of I13522 were single colony PCRed (see gel image below) and used as inoculate for 5 mL of LB for later plasmid isolation and -80 °C stocks.

1% Agarose gel of scPCR product:

1 % agarose of colony PCR. Gel runned 1 hour at 100 V. Of all samples 5 μL + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane descriptions:

# Description Expected Length (bp) Primers Status
M1 SmartLadder marker n/a n/a n/a
1 transformant #1 of J23100 G00100 + G00101
2 transformant #2 of J23100 G00100 + G00101
3 transformant #1 of I13522 G00100 + G00101
M2 EZ Load Ladder (5 μL) n/a n/a n/a


From the gel it can be concluded that the transformants carry the desired plasmids with the proper BioBrink inserts.