Team:TU Delft/19 July 2010 content

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(Characterization of Anderson RBS sequences)
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In order to continue progress made on the third method, described in the blog of [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=14_July_2010 July 14th] for the assembly of the reference construct J23100-B0032-E0040-B0015, the following [[Team:TU_Delft/protocols/restriction_enzyme_digestion_40| Digestions]] were performed:
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In order to continue progress made on the fourth method, described in the blog of [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=14_July_2010 July 14th] for the assembly of the reference construct J23100-B0032-E0040-B0015, the following [[Team:TU_Delft/protocols/restriction_enzyme_digestion_40| Digestions]] were performed:
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To all samples with an end volume of 25 μL, 2.5 μL ligase buffer was added.
To all samples with an end volume of 25 μL, 2.5 μL ligase buffer was added.
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Note: The above method is not exactly the proposed method #3, but a variation without PCR amplification of E0240. This is less elegant; while a re-ligation of the J61002 plasmid will result in colonies with red fluorescence, a re-ligation of the pSB1A2 plasmid containing E0240 cannot easily be screened against (or selected against as both contain ampicillin markers).
 
In the likelihood that the abovementioned method will not yield desired transformants, a [[Team:TU_Delft/protocols/PCR|PCR amplification]] of E0240 was performed in three-fold. The resulting product over 1% agarose gel can be seen below. The PCR amplification product was subsequently digested using XbaI and PstI for later insertion into previously digested (SpeI and PstI) J23100(in J61002).
In the likelihood that the abovementioned method will not yield desired transformants, a [[Team:TU_Delft/protocols/PCR|PCR amplification]] of E0240 was performed in three-fold. The resulting product over 1% agarose gel can be seen below. The PCR amplification product was subsequently digested using XbaI and PstI for later insertion into previously digested (SpeI and PstI) J23100(in J61002).

Revision as of 14:59, 12 August 2010

Contents

Forbidden word

Today we chose a word that we were not allowed to say all day. The word was pipet. It turned out to be hard not to use this word. All kind of funny definitions were used to avoid the word pipet. Still some persons accidentally said the word and have to do the dishes tomorrow.

Lab work

Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing

The frozen pellet of last week were isolated using Birnboim protocol.

The following plasmid concentrations were obtained using the nanodrop:

BioBrick Composed of Concentration (ng/μL)
K398328T AlkS and B0015 145.01
K398407T PhPFDa and B0032 37.8
Control pSB1T3 and B0015 71.65
K398300C AlkS 24.54
Control pSB1C3 93.86
K398200C AlnA 4.23
K398000C LadA 148.14
K398002C RubA3 119.85
K398201C OprG 161.28
K398400C PhPFDa 163.33

Because not all ligation mixes resulted in transformants were repeated the transformation with the overnight ligated reactions.

Alkane degradation

Biobricks in production: K398007T, K398008T, K398009T, K398010T, K398016T, K398017T & K398018T

>Digestion

Digestion reactions:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 2 μg J61100 EcoRI SpeI 2 (BioLabs) ‘E–J61100–S’
2 2 μg J61100 EcoRI SpeI 2 (BioLabs) ‘E–J61100–S’
3 1 μg rubR EcoRI SpeI 2 (BioLabs) ‘E–rubR–S’
4 1 μg J61101 EcoRI SpeI 2 (BioLabs) ‘E–J61101–S’
5 1 μg J61107 EcoRI SpeI 2 (BioLabs) ‘E–J61107–S’
6 1 μg alkB2 Xbal PstI 2 (BioLabs) ‘X–alkB2–P’
7 1 μg rubA3 Xbal PstI 2 (BioLabs) ‘X–rubA3–P’
8 1 μg rubA4 Xbal PstI 2 (BioLabs) ‘X–rubA4–P’
9 1 μg B0015 Xbal PstI 2 (BioLabs) ‘X–B0015–P’
10 1 μg ladA Xbal PstI 2 (BioLabs) ‘X–ladA–P’
11 1 μg ADH Xbal PstI 2 (BioLabs) ‘X-ADH–P’
12 1 μg ALDH Xbal PstI 2 (BioLabs) ‘X–ALDH–P’
13 3 μg pSB1T3 EcoRI PstI 2 (BioLabs) ‘E–linear pSB1T3–P’


Results of the digestion on 1% agarose gel

1% agarose of digestion check. Gel runned at 100V for 1 hour. Of all samples 5 μL was loaded with 1 μL loadingbuffer. 5 μL was loaded of marker

Lane description:

# Description Expected Length (bp) Status Remarks
1 Easy load marker n/a n/a
2 J61100 + EcoRI + SpeI 78, 2056 ? Not visible (too small)
3 J61100 + EcoRI + SpeI 78, 2056 ? Not visible (too small)
4 rubR + EcoRI + SpeI 1227, 2326
5 J61101 + EcoRI + SpeI 78, 2056 ? Not visible (too small)
6 J61107 + EcoRI + SpeI 78, 2056 ? Not visible (too small)
7 pSB1A2-J23100 + SpeI + PstI 2110 low concentration
8 alkB2 + XbaI + PstI 1255, 2411
9 rubA3 + XbaI + PstI 198, 2409 ? Not visible on picture, but was vaguely seen
10 rubA4 + XbaI + PstI 207, 2409 ? Not visible on picture, but was vaguely seen
11 B0015 + XbaI + PstI 155, 3163 ? Not visible (too small)
12 ladA + XbaI + PstI 1350, 2915
13 ADH + XbaI + PstI 769, 2411
14 ALDH + XbaI + PstI 1521, 1618, 705 Originally in a Kan backbone which gives an extra fragment of 800 bp when cut, rest of the backbone and the fragment both ~ 1500, so overlap
15 E0240 + XbaI + PstI 902, 2053
16 pSB1T3 + EcoRI + PstI 2422, 1350
17 SmartLadder n/a n/a

Ligation

The digestion products were ligated over night:

# BioBrick Fragment 1 Fragment 2 Recipient vector Final volume
1 K398007T 6 μL ‘E-J61100-S’ 12 μL ‘X-alkB2-P’ 1.3 μL ‘E-pSB1T3-P’ 25 μL
2 K398008T 6 μL ‘E-J61100-S’ 11 μL ‘X-rubA3-P’ 1.3 μL ‘E-pSB1T3-P’ 25 μL
3 K398009T 6 μL ‘E-J61100-S’ 12.5 μL ‘X-rubA4-P’ 1.3 μL ‘E-pSB1T3-P’ 25 μL
4 K398010T 10 μL ‘E-rubR-S’ 11 μL ‘X-B0015-P’ 1.3 μL ‘E-pSB1T3-P’ 25.8 μL
5 K398016T 6 μL ‘E-J61100-S’ 12 μL ‘X-ladA-P’ 1.3 μL ‘E-pSB1T3-P’ 25 μL
6 K398017T 12.5 μL ‘E-J61101-S’ 11 μL ‘X-ADH-P’ 1.3 μL ‘E-pSB1T3-P’ 30 μL*
7 K398018T 12.5 μL ‘E-J61107-S’ 11.5 μL ‘X-ALDH-P’ 1.3 μL ‘E-pSB1T3-P’ 30 μL*
8 n/a 10.5 μL ‘X-E0240-P’ n/a 4 μL ‘S-pSB1A2-J23100-P’ 25 μL

To all samples with an end volume of 25μL (and 25.8μL), 2.5 μL Ligase buffer was added. For those with an end volume of 30μL (indicated by an *) 3.0μL Ligase buffer was added.

Emulsifier

Since last weeks attempt to construct the inducible protomer R0011 with RBS B0032 failed, we decided to take a different approach. Instead of doing the standard 2 parts assembly, we amplify the promoter by PCR digest it and ligate in the plasmid that contains the RBS which has only be cut open at the left side. By doing so we do not risk losing the super small DNA fragments like the RBS. The problem is that we cannot use antibiotics selection. So we need to determine that by colony PCR and sequencing.

PCR Amplification

R0011 was amplified with the universal primers G00100 and G00101. The product was put on 1% agarose gel:

1% agarose of PCR. Gel runned at 100 V for 1 hour. Of all samples 10 μL was loaded with 2 μL loadingbuffer. 5 μL was loaded of marker

Lane description:

# Description Expected length (bp) Primers Status Remarks
M1 BioRad EZ Ladder n/a n/a n/a
1 PCR Product of R0011 293 G00100 + G00101 R0011 itself is just 55 bp, but the flanking primer regions are about 100 bp each

The PCR band is about 300 bps long. R0011 itself is just 55 bp, but the flanking primer regions are about 100 bp each.

Digestion

The PCR product of promotor R0011 was cut with EcoRI and SpeI and the RBS containing plasmid has been cut open with EcoRI and Xbal for 2.5 hours at 37 °C. This deviates from the standard biobrick assembly, thus is not completely as described in our digestion protocol.

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1 μg B0032 EcoRI Xbal 2 (BioLabs) ‘X–B0032 pSB1A2–E’
2 30 μL PCR product of R0011 EcoRI SpeI 2 (BioLabs) ‘E-R0011-S’

Ligation

The digestion products were ligated over night:

# BioBrick Fragment 1 Fragment 2
1 K398202 5 μL ‘X–B0032 pSB1A2–E’ 10 μL ‘E-R0011-S’

Hopefully this will lead to 'E-X-R0011-B0032-S-P' in the B0032 plasmid backbone with Ampicillin resistance marker.

Competent cells

Top10 and DH5α cells were cultivated for making competent cells

Characterization of Anderson RBS sequences

Assembly of reference construct

Friday's ligation products, ligation control and digestion control were transformed into chemically competent Top10 cells and incubated on ampicillin plates.

# BioBrick Plasmid
T1 K081005-I13401 pSB1A2
T2 K081005-I13401 pSB1A2
T3 I13401 (ligation control) pSB1AK2
T4 'E-pSB1A2-I13401-X' (digest control) pSB1AK2
T5 MilliQ -

Similarly a third attempt was made at transforming K081005, this time into commercial chemically competent cells.

# BioBrick Plasmid
T6 K081005 pSB1A2

In order to continue progress made on the fourth method, described in the blog of July 14th for the assembly of the reference construct J23100-B0032-E0040-B0015, the following Digestions were performed:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1 μg J23100 SpeI PstI 2 (BioLabs) ‘S–J23100 in J61002–P’
2 1 μg E0240 XbaI PstI 2 (BioLabs) ‘X–E0240–P’

After enzyme inactivation, the digestion products were set for ligation overnight.

# BioBrick Fragment 1 Fragment 2 Recipient vector Final volume
1 K173000 10.5 μL ‘X-E0240-P’ - 4 μL ‘S-pSB1A2-J23100-P’ 25 μL

To all samples with an end volume of 25 μL, 2.5 μL ligase buffer was added.

In the likelihood that the abovementioned method will not yield desired transformants, a PCR amplification of E0240 was performed in three-fold. The resulting product over 1% agarose gel can be seen below. The PCR amplification product was subsequently digested using XbaI and PstI for later insertion into previously digested (SpeI and PstI) J23100(in J61002).

1% Agarose gel of E0240 PCR product:

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

Lane descriptions:

# Description Expected Length (bp) Primers Status Remarks
1 SmartLadder marker n/a n/a n/a
2 PCR product of E0240 #1 1114 G00100 + G00101
3 PCR product of E0240 #2 1114 G00100 + G00101
4 PCR product of E0240 #3 1114 G00100 + G00101