Team:TU Delft/19 July 2010 content

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(Characterization of Anderson RBS sequences)
(Characterization of Anderson RBS sequences)
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==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
<h5>Assembly of reference construct</h5>
<h5>Assembly of reference construct</h5>
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[http://2010.igem.org/Team:TU_Delft#/blog?blog=16_July_2010 Friday's] ligation products were [http://2010.igem.org/Team:TU_Delft/protocols/transformation transformed] into chemically competent TOP10 cells. Similarly a third attempt was made at transforming K081005, this time into commercial chemically competent cells.
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[http://2010.igem.org/Team:TU_Delft#/blog?blog=16_July_2010 Friday's] ligation products were [http://2010.igem.org/Team:TU_Delft/protocols/transformation transformed] into chemically competent TOP10 cells and incubated on AMP plates. Similarly a third attempt was made at transforming K081005, this time into commercial chemically competent cells.
In order to continue progress made on the third method, described in the blog of [http://2010.igem.org/Team:TU_Delft#/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:
In order to continue progress made on the third method, described in the blog of [http://2010.igem.org/Team:TU_Delft#/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:

Revision as of 22:06, 20 July 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:

BioBrick Composed of Concentration (ng/μL)

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:

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


Results of the digestion on 1% agarose gel

Digestion check


Lane description:

# Description Expected Length (bp) Remarks
1 Easy load marker (5 μL) n/a
2 ‘E – J61100 – S’ 78 Not visible (too small)
3 ‘E – J61100 – S’ 78 Not visible (too small)
4 ‘E – rubR– S’ 1227 Visible
5 ‘E – J61101– S’ 78 Not visible (too small)
6 ‘E – J61107– S’ 78 Not visible (too small)
7 ‘S-pSB1A2-J23100-P’ 2110 Visible (low concentration)
8 ‘X – alkB2 – P’ 1263 Visible
9 ‘X – rubA3 – P’ 198 Not visible on picture, but was vaguely seen
10 ‘X – rubA4 – P’ 207 Not visible on picture, but was vaguely seen
11 ‘X – B0015 – P’ 155 Not visible (too small)
12 ‘X – ladA – P’ 1350 Clearly visible
13 ‘X – ADH – P’ 777 Visible
14 ‘X – ALDH – P’ 1521 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 ‘X – E0240 – P’ 876 Visible
16 ‘E-pSB1T3-P’ 2422 Visible
17 Smartladder (5μL) n/a

Of all samples 5 μL was loaded with 1 μL loadingbuffer (except the ladders)

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 gel

Lane description:

# Description
1 BioRad EZ Ladder (5 μL)
2 PCR Product of R0011 (10 μL + 2 μL Loading buffer)

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.

# Digestion reaction Used Buffer Needed fragment
1 1 μg B0032 + EcoRI + Xbal: Buffer 2 (BioLabs) ‘X – B0032 - Plasmid backbone – E’
2 30 μL PCR product of R0011 + EcoRI + SpeI Buffer 2 (BioLabs) ‘E - R0011 - S’

Ligation

The digestion products were ligated over night:

# Ligation reaction
1 5 μL B0032 + 10 μL PCR Product of R0011

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 were transformed into chemically competent TOP10 cells and incubated on AMP plates. Similarly a third attempt was made at transforming K081005, this time into commercial chemically competent cells.

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

# Digestion reaction Used Buffer desired fragment
1 1 μg J23100 + SpeI + PstI Buffer 2 (BioLabs) ‘S – J23100(J61002) – P’
2 1 μg E0240 + XbaI + PstI Buffer 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’ None 4 μL ‘S-pSB1A2-J23100-P’ 25 μL

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

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 AMP markers).

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).

Images of the day

1% Agarose gel of E0240 PCR product:

1% Agarose gel of PCR product

Lane descriptions:

# Description Expected Length (bp)
1 SmartLadder marker (5 μL) Various
2 PCR product of E0240 #1 (5 μL + 1 μL loadingbuffer) 1114
3 PCR product of E0240 #2 (5 μL + 1 μL loadingbuffer) 1114
4 PCR product of E0240 #3 (5 μL + 1 μL loadingbuffer) 1114