Team:TU Delft/20 July 2010 content

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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

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: J23100-B0030-I13401 G00101 + G00101
3 transformant #2 of ligation mix: J23100-B0030-I13401 G00101 + G00101
4 transformant #3 of ligation mix: J23100-B0030-I13401 G00101 + G00101
5 transformant #4 of ligation mix: J23100-B0030-I13401 G00101 + G00101
6 transformant #5 of ligation mix: J23100-B0030-I13401 G00101 + G00101
7 transformant #1 of ligation mix: I13401 ligation control G00101 + G00101
8 transformant #2 of ligation mix: I13401 ligation control G00101 + G00101
9 transformant #3 of ligation mix: I13401 ligation control G00101 + G00101
10 transformant #1 of ligation mix: I13401 digestion control G00101 + G00101
11 transformant #2 of ligation mix: I13401 digestion control G00101 + G00101
12 transformant #1 of K081005 G00101 + G00101
13 transformant #2 of K081005 G00101 + G00101
14 transformant #3 of K081005 G00101 + G00101
15 BioRad EZ Load n/a n/a n/a
16 PCR product of E0240, XbaI and PstI None
17 PCR product of E0240, XbaI and PstI None
18 PCR product of E0240, XbaI and PstI None


From the gel it could be determined that there were no positive colonies containing K081005-I13401 in pSB1AK2. The ligation controls show the presence of re-ligated I13401 plasmids. 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 ligation control colonies (lanes 7 and 9) and that of the PCR product of K081005. An explanation for this could be that I13401 is excised from its plasmid due to an additional XbaI site located in the plasmid and K081005 ligated in its place. We tested this hypothesis by digesting I13401-pSB1A2 with XbaI and running the product on gel.

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.