Team:TU Delft/20 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Assembly of reference construct)
(Assembly of reference construct)
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We tested hypothesis I by [http://2010.igem.org/Team:TU_Delft#page=protocols/restriction_enzyme_digestion digesting] I13401 in pSB1AK2 with XbaI and running the product on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel1% agarose gel]:
We tested hypothesis I by [http://2010.igem.org/Team:TU_Delft#page=protocols/restriction_enzyme_digestion digesting] I13401 in pSB1AK2 with XbaI and running the product on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel1% agarose gel]:
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[[Image:TU_Delft_Digestion_XbaI_I13401.png|500px|thumb|left|1% agarose of I13401 digestion with XbaI. Gel runned at 100V for 1 hour. 5 μL was loaded of marker]]
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|-
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|1
|1
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|SmartLadder
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|None 
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|2 (Biolabs)
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|✓
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|+
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|-
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|2
|I13401-pSB1AK2
|I13401-pSB1AK2
|XbaI   
|XbaI   
-
|2 (Biolabs)
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|
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|✓
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|+
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|-
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|3
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|BioRad EZ Load
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|None 
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|
|✓
|✓
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|+
|}
|}
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[[Image:TU_Delft_Digestion_XbaI_I13401.png|500px|thumb|left|1% agarose of I13401 digestion with XbaI. Gel runned at 100V for 1 hour. 5 μL was loaded of marker]]
The gel of the digestion product showed the expected digested plasmid length and lacked bands that could indicate an extra XbaI site.
The gel of the digestion product showed the expected digested plasmid length and lacked bands that could indicate an extra XbaI site.

Revision as of 09:52, 13 August 2010

Contents

Juggling an Diabolo tricks!

From all the pipetting that we do in the lab, our fingers and wrists are a bit tense. After some hard thinking, we found a perfect way to relax them…..by juggling and performing diabolo tricks! So now, in between the incubation times of our experiments the juggle balls are flying around in our iGEM room.

Lab work

Competent cells

Eva made another batch of competent cells

Alkane degradation

BioBrick production continued

A transformation of yesterday's ligation products was done in TOP10 competent cells, and grown on LB-Tetracycline plates overnight. Hopefully we will have some colonies tomorrow.


Salt tolerance

Time to get cracking, so bbc1 1T3 and B0015 were restricted using the scheme shown below. These were then ligated to one another in the third scheme.

# Sample added Volume added (µl)
1 bbc1 10
Ecro R1 1
Xba1 1
Buffer 2 (biolabs) 2
Water 4
BSA 2
2 B0015 10
Spe1 1
Pst1 1
Buffer 2 (biolabs) 2
Water 4
BSA 2
3 psb 1T3 1
Ecro R1 1
Pst1 1
Buffer 3 (biolabs) 2
Water 6


The Ligation (as shown below) was split into two tubes with 25 µl and ligated overnight.

# Sample added Volume added (µl)
1 psB1T3 2
bbc1 18
B0015 18
Ligase buffer 5
T4 DNA Ligase 2
Water 5

Characterization of Anderson RBS sequences

Assembly of reference construct

Method 1

Single colony PCRs were performed on the transformants obtained yesterday. These were loaded onto 1% agarose gel (see gel below)

1% agarose of colony 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 description:

# Description Expected Length (bp) Primers Status Remarks
1 SmartLadder n/a n/a n/a
2 transformant #1 of ligation mix: E-K081005-S + E-pSB1AK2-I13401-X 1239 G00101 + G00101
3 transformant #2 of ligation mix: E-K081005-S + E-pSB1AK2-I13401-X 1239 G00101 + G00101
4 transformant #3 of ligation mix: E-K081005-S + E-pSB1AK2-I13401-X 1239 G00101 + G00101
5 transformant #4 of ligation mix: E-K081005-S + E-pSB1AK2-I13401-X 1239 G00101 + G00101
6 transformant #5 of ligation mix: E-K081005-S + E-pSB1AK2-I13401-X 1239 G00101 + G00101
7 transformant #1 of ligation mix: E-pSB1AK2-I13401-X (ligation control) 1173 G00101 + G00101
8 transformant #2 of ligation mix: E-pSB1AK2-I13401-X (ligation control) 1173 G00101 + G00101
9 transformant #3 of ligation mix: E-pSB1AK2-I13401-X (ligation control) 1173 G00101 + G00101
10 transformant #1 of digestion mix: E-pSB1AK2-I13401-X (digestion control) 1173 G00101 + G00101
11 transformant #2 of digestion mix: E-pSB1AK2-I13401-X (digestion control) 1173 G00101 + G00101
12 transformant #1 of K081005 in pSB1A2 296 G00101 + G00101
13 transformant #2 of K081005 in pSB1A2 296 G00101 + G00101
14 transformant #3 of K081005 in pSB1A2 296 G00101 + G00101
15 BioRad EZ Load n/a n/a n/a
16 Digested PCR product of E0240, XbaI and PstI 898 None
17 Digested PCR product of E0240, XbaI and PstI 898 None
18 Digested PCR product of E0240, XbaI and PstI 898 None


From the gel it could be determined that there weren't any positive colonies containing K081005-I13401 in pSB1AK2. The ligation controls show the presence of back-ligated E-pSB1AK2-I13401-X. The digestion controls show colonies containing I13401 in pSB1AK2, which is attributed to an incomplete digestion. It is interesting to note the similarity of the length of the bands of the inserts belonging to the transformants (lanes 1 through 5) and that of the PCR product of K081005. Hypothesis I: An explanation for this could be that I13401 is excised from its plasmid due to an additional XbaI site located in the plasmid with subsequent ligation of K081005 in its place. Hypothesis II: Another explanation could be that the template plasmid used for the amplification of K081005 was transformed.

Hypothesis II

We first tested hypothesis II by plating out yesterday's transformants onto kanamycin plates. Growth on kanamycin plates would indicate the presence of the pSB1AK2 plasmid and thus that the cells did not contain the PCR template plasmid (K081005), as this plasmid backbone only contains the ampicillin resistance marker.

Hypothesis I

We tested hypothesis I by digesting I13401 in pSB1AK2 with XbaI and running the product on agarose gel:

1% agarose of I13401 digestion with XbaI. Gel runned at 100V for 1 hour. 5 μL was loaded of marker
# Sample Enzyme 1 Buffer BSA
1 SmartLadder None 2 (Biolabs)
2 I13401-pSB1AK2 XbaI
3 BioRad EZ Load None
1% agarose of I13401 digestion with XbaI. Gel runned at 100V for 1 hour. 5 μL was loaded of marker

The gel of the digestion product showed the expected digested plasmid length and lacked bands that could indicate an extra XbaI site.

The fact that some colonies of the ligation control (lanes 7 and 9) also appear to show bands similar to the PCR size of K081005 further indicates that hypothesis I is faulty, seeing as these ligation mixes never got in contact with K081005.

Method 3

Yesterday's digested PCR product of E0240 was ligated into S-J23100-pSB1A2-P.

Beach

After a hard day working in the lab, we went to the beach in Scheveningen for a nice walk and to watch the sunset.