Team:TU Delft/14 July 2010 content

From 2010.igem.org

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(Lab work)
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==Lab work==
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=Lab work=
<h4>Ordered DNA</h4>  
<h4>Ordered DNA</h4>  
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'''lane 16'''
'''lane 16'''
Expected length of J04450 is 1090 bp (without flanking primer regions). On gel one band is visible in lane 16 at height 1400 bp.
Expected length of J04450 is 1090 bp (without flanking primer regions). On gel one band is visible in lane 16 at height 1400 bp.
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==Characterization of Anderson RBS sequences==
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The first results are in from [http://2010.igem.org/Team:TU_Delft#/blog?blog=13_July_2010 last night's] measurements!
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The results showed unexpected OD600 curves which were attributed to excessive evaporation of water from the wells. Time for attempt#2 (with evaporation restrictions): The TOP 10 strains carrying K398500, K398501, K398502, K398503, K398504 and I13401 were cultured overnight in 5 mL LB medium (37℃, 200 rpm).
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In order to properly characterize the Anderson RBS sequences a 'reference' RBS will be run in parallel with the fluorescence measurements of the Anderson RBS sequences. Well characterized and standardized RBSs are the community RBSs, especially B0030, B0032 and B0034. A useful construct which could be used as the reference in these experiments has the form of: J23100 - B0030 or B0032 or B0034 - GFP - dT. As these constructs are (unfortunately) not readily available from the Spring 2010 distribution plates, they will have to be created:
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1. The simplest method involves PCR amplifying [http://partsregistry.org/Part:BBa_K081005 K081005], digesting with EcoRI and SpeI and ligation into a pre-cut (EcoRI and XbaI) [http://partsregistry.org/Part:BBa_I13401 I13401] in pSB1A2 (which we have in stock).
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2. The second method involves the transformation of [http://partsregistry.org/Part:BBa_K081005 K081005] into chemically competent TOP10 cells, overnight culturing and plasmid isolation. The obtained K081005 in pSB1A2 can be cut by SpeI and PstI and ligated to the PCR amplified and digested (XbaI and PstI) I13401. This is [http://2010.igem.org/Team:TU_Delft#/blog?blog=6_July_2010 analogous] to the method used for obtaining the Anderson RBS constructs K398500-K398504.
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3. The third method involves transforming [http://partsregistry.org/Part:BBa_J23100 J23100] into chemically competent TOP10 cells, overnight culturing and plasmid isolation. The obtained J23100 in J61002 can be cut by SpeI and PstI and ligated to the PCR amplified and digested (XbaI and PstI) [http://partsregistry.org/Part:BBa_E0240 E0240].
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Thus, BioBricks K081005 and J23100 were [[Team:TU_Delft/protocols/transformation|transformed]] into chemically competent TOP10 cells and K081005 was PCR amplified []

Revision as of 21:15, 19 July 2010

Contents

Lab work

Ordered DNA

Yesterdays digestions were tested on gel. At the same time the digestion products were ligated at 16 °C:

# Ligation reaction
1 8 μL alkB2 + 1 μL pSB1C3
2 8 μL rubA3 + 1 μL pSB1C3
3 8 μL ladA + 1 μL pSB1C3
4 8 μL ADH + 1 μL pSB1C3
5 8 μL AlnA + 1 μL pSB1C3
6 8 μL OprG + 1 μL pSB1C3
7 8 μL AlkS + 1 μL pSB1C3
8 8 μL PalkB + 1 μL pSB1C3
9 8 μL PalkS12 + 1 μL pSB1C3
10 8 μL PhPFDα + 1 μL pSB1C3
11 8 μL PhPFDβ + 1 μL pSB1C3
12 1 μL pSB1C3 (negative control)

After 4 hour the 10 μL of the ligation mixes were transformed in Top10 competent cells. The rest of the ligation mix was overnight incubated at 16 °C.

Solvent Tolerance and Hydrocarbon Sensing

Ligation of yesterdays digestion reactions:

# Ligation reaction
1 8 μL AlkS + 8 μL E0422 + 1 μL pSB1T3
2 8 μL AlkS + 8 μL B0015 + 1 μL pSB1T3
3 8 μL PhPFDα + 8 μL B0032 + 1 μL pSB1T3
4 8 μL PhPFDβ + 8 μL B0015 + 1 μL pSB1T3
5 8 μL B0015 + 1 μL pSB1T3 (negative control)
6 1 μL pSB1T3 (negative control)

After 4 hour the 10 μL of the ligation mixes were transformed in Top10 competent cells. The rest of the ligation mix was overnight incubated at 16 °C.

Emulsifier

The transformations of yesterday containing the different ligation reactions gave colonies. All plates contained red and white colonies. This is probably due to the fact that we did not purified the backbone before ligation. So some plasmids reassembled. Pieter picked 5 colonies of every plate (no red ones) and 1 from the control plate (a red colony) and performed a colony PCR to check which colonies contained the right insert. At the same time he cultivated the colonies in 5 mL LB medium containing the 15 μg/mL name Tetracycline to grow over night at 37 °C.

The PCR products were run on gel a 1% agarose gel.

Colony PCR Products

Lane Description

# Description Expected lenght (bp)
0 SmartLadder (5 μL)
1 Transformant 1 of Ligation mix: R0011 + B0032 + pSB1T3 (10 μL + 2 μL loadingbuffer)
2 Transformant 2 of Ligation mix: R0011 + B0032 + pSB1T3 (10 μL + 2 μL loadingbuffer)
3 Transformant 3 of Ligation mix: R0011 + B0032 + pSB1T3 (10 μL + 2 μL loadingbuffer)
4 Transformant 4 of Ligation mix: R0011 + B0032 + pSB1T3 (10 μL + 2 μL loadingbuffer)
5 Transformant 5 of Ligation mix: R0011 + B0032 + pSB1T3 (10 μL + 2 μL loadingbuffer)
6 Transformant 6 of Ligation mix: AlnA + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
7 Transformant 7 of Ligation mix: AlnA + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
8 Transformant 8 of Ligation mix: AlnA + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
9 Transformant 9 of Ligation mix: AlnA + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
10 Transformant 10 of Ligation mix: AlnA + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
11 Transformant 11 of Ligation mix: OprG + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
12 Transformant 12 of Ligation mix: OprG + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
13 Transformant 13 of Ligation mix: OprG + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
14 Transformant 14 of Ligation mix: OprG + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
15 Transformant 15 of Ligation mix: OprG + B0015 + pSB1T3 (10 μL + 2 μL loadingbuffer)
16 Transformant 16 of Ligation mix: pSB1T3 (10 μL + 2 μL loadingbuffer)

lane 1-5 Expected length of R0011 + B0032 = 55 + 13 = 83 bp (without flanking primer regions). On gel there are only bands poorly visible at height of 1500 bp. So the transformation of the Promotor and RBS has failed.

lane 6-10 Expected lenght of OprG + B0015 = 744 + 130 = 870 bp (without flanking primer regions). On gel bands are visible of about 1000 bp in lanes 6,7 and 10. The bands just a little lower are unidentified. The OprG with terminator is probably in cultures 6 and 7.

Lane 11-15 Expected length of AlnA + B0015 = 1107 + 130 = 1237 bp (without flanking primer regions). On gel bands are visible of about 1400 bp in lanes 11,13 and 15. Since bands of AlnA with terminator and J04450 are about the same size, it is not clear whether this transformation succeeded. Tomorrow I will check the isolated plasmids on gel with a characteristic digesition reaction.

lane 16 Expected length of J04450 is 1090 bp (without flanking primer regions). On gel one band is visible in lane 16 at height 1400 bp.

Characterization of Anderson RBS sequences

The first results are in from last night's measurements! The results showed unexpected OD600 curves which were attributed to excessive evaporation of water from the wells. Time for attempt#2 (with evaporation restrictions): The TOP 10 strains carrying K398500, K398501, K398502, K398503, K398504 and I13401 were cultured overnight in 5 mL LB medium (37℃, 200 rpm).

In order to properly characterize the Anderson RBS sequences a 'reference' RBS will be run in parallel with the fluorescence measurements of the Anderson RBS sequences. Well characterized and standardized RBSs are the community RBSs, especially B0030, B0032 and B0034. A useful construct which could be used as the reference in these experiments has the form of: J23100 - B0030 or B0032 or B0034 - GFP - dT. As these constructs are (unfortunately) not readily available from the Spring 2010 distribution plates, they will have to be created:

1. The simplest method involves PCR amplifying K081005, digesting with EcoRI and SpeI and ligation into a pre-cut (EcoRI and XbaI) I13401 in pSB1A2 (which we have in stock).

2. The second method involves the transformation of K081005 into chemically competent TOP10 cells, overnight culturing and plasmid isolation. The obtained K081005 in pSB1A2 can be cut by SpeI and PstI and ligated to the PCR amplified and digested (XbaI and PstI) I13401. This is analogous to the method used for obtaining the Anderson RBS constructs K398500-K398504.

3. The third method involves transforming J23100 into chemically competent TOP10 cells, overnight culturing and plasmid isolation. The obtained J23100 in J61002 can be cut by SpeI and PstI and ligated to the PCR amplified and digested (XbaI and PstI) E0240.

Thus, BioBricks K081005 and J23100 were transformed into chemically competent TOP10 cells and K081005 was PCR amplified []