"

From 2010.igem.org

Contents 1 Andreas 1.1 Testing LB agar plates with antibiotics 1.2 Transformations 1.3 Isolation of requested parts 2 Hassan 2.1 Continuation from yesterday 2.2 Reference 2.3 TODO 3 Johan 3.1 Transformation 3.2 Gel 3.3 Gel purification

Andreas

Testing LB agar plates with antibiotics

Results from 1/7 platings with non-transformed Top10 cells

• 50 ug/ml Km: No growth
• 50 ug/ml Amp & 25 ug/ml Cm: No growth
• 50 ug/ml Amp & 25 ug/ml Km: No growth

Transformations

Results from 1/7 transformations

Top10

• pSB1A3.BBa_J04450: Growth (red colonies)
• pSB1C3.BBa_J04450: Growth (red colonies)
• pSB1K3.BBa_J04450: Growth (red colonies)
• pSB1AC3.BBa_J04450: Growth (red colonies)
• pSB1AK3.BBa_J04450: Growth (red colonies)

Colonies inoculated in 3 ml LB with appropriate antibiotics and grown in 37°C, 250 rpm for 6 hours, and 15% glycerol stocks were prepared. No glycerol stock was prepared for pSB1K3, as the screw cap came off in the shake incubator; this will be prepared later.

Plates photographed and stored in 4°C.

BL21(DE3)

• pEX.SOD: Growth
• pEX.yCCS: Growth
• pMA.BBa_K157011: Growth

Plates photographed and stored in 4°C.

Isolation of requested parts

Results from 1/7 platings

• pMA.BBa_J18930 (mCerulean; CFP): Growth
• pMA.BBa_J18931 (mCitrine; YFP): Growth
• pMA.BBa_J18932 (mCherry; RFP): Growth

Single colonies inoculated in 3 ml LB with 100 ug/ml Amp and grown in 37°C, 250 rpm for 6 hours. Glycerol stocks were then prepared.

Plates photographed and stored in 4°C.

Hassan

Continuation from yesterday

1. Prieto et. al. (2008) Used KEGG for their study of functional interaction, used method: "The data used in this work corresponds to a set of human genome-wide expression microarrays hybridized with mRNA samples coming from different human tissues, glands or organs from healthy normal individuals." With notes from yesterday about this article, I think these information could help us understand which genes and prot. interaction to trust.
   
2. De Las Rivas et al 2010

• "As discussed previously (Joel P. Mackay et al 2007 ^[1],Andrew Chatr-aryamontri et al 2008^[2] ), the issue of whether two proteins share a “functional contact” is quite distinct from the question of whether the same two proteins interact directly with each other. Any protein in the ribosome or in the basal transcriptional apparatus shares a functional contact with the other proteins in the complex, but certainly not all the proteins in the particular complex interact."
  
• "Another essential element for defining PPIs is the biological context. Not all possible interactions will occur in any cell at any time. Instead, interactions depend on cell type, cell cycle phase and state, developmental stage, environmental conditions, protein modifications (e.g., phosphorylation), presence of cofactors, and presence of other binding partners."
  
• "The techniques that measure direct physical interactions between protein pairs are “binary” methods, while the techniques that measure physical interactions among groups of proteins, without pairwise determination of protein partners, are “co-complex” methods"
  

Reference

• Joel P. Mackay et al 2007 ^[1]
• Andrew Chatr-aryamontri et al 2008^[2]

TODO

Read [3] was a bad TODO!

Johan

Transformation

bFGF (80 ng/µl) into competent BL21 cells.

5 µl of plasmid used, 50 µl of sample spreaded into plates

Gel

Rest of pEX, was then cutted.

Gel purification

(QIAGEN - QIAEX II Agarose Extraction Protocol)

I did: 8 pEX samples in freezer from yesterday, each having gel from two lanes (250-300 mg).

• 2) 0,75 ml Buffer QX1 + 0,5 ml H2O
• 3) 30 µl QIAEX II
• 9) Incubate at 50 °C for 5 min
• 4) 10 min incubation with vortexing didnt solubilize all agarose.
• 10 min incubation extra without vortexing solubilized 4 of the 8 samples.
• For the other 4 samples, 500 µl more Buffer QX1 and 10 µl more QIAEX II and 5 min incubation, and all was solubilized.
• Step 11) was discarded.

All samples was added to one tube, 180 µl.

Absorbance: 528 / 514 (0,103 0,060 0,014).