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From 2010.igem.org

To synthesize the minimal chromosome, we selected some essential genes and got each gene sequence from NCBI Genome database. Replication origin(including rctA, rctB) , parA, parB, parS, dif and related promoter. Designing primer including BioBrick prefix and suffix using GinkoPrimer tool.

parS and dif is very short sequence DNA. So we decided to make these genes using PCR primer including BioBrick prefix and suffix.

   parS, ParB protein binding sites have 9 different sequences and positions in V.cholerae chrII each other.  So we take consensus sequence of 9 parS sequences.

 To checking the V.cholerae origin is working well, we first used BioBrick base vector BBa_I51020. But there is ccdb positive selection marker that killed the host. So we decide to re-design the pSB1C3 BioBrick vector using PCR. And we got the re-designed vector, Cn. The Cn has 2 NheI restriction enzyme sites instead rep protein coding gene. XbaI/SpeI can make mixed sequence with NheI.

EcoRI and PstI sites are located in parB gene. Because of this, we should cut parB to XbaI/SpeI. It could be inserted into Cn, NheI site. But we have to consider gene arrangement.

DNA Amplification
Concentration of template DNA and primer. Add distilled water to AccuPower™ HF PCR PreMix(Bioneer) tubes to a total volume of 20㎕.

Step	Temperature	Time	Number of cycles
Initial Denaturation	94℃	5min	1cycle
Denaturation	94℃	1min	The cycle number is dependent on the amount of template DNA.
Annealing	68℃	1min
Extension	72℃	1min/kb
Final Extension	72℃	5min	1cycle

In case of long PCR It is recommended to use two-step PCR methods.

Step	Temperature	Time	Number of cycles
Initial Denaturation	94℃	5min	1cycle
Denaturation	94℃	20sec	The cycle number is dependent on the amount of template DNA.
Annealing/Extension	68℃	1min
Final Extension	72℃	5min	1cycle

Plasmid Mini Extraction
The overall principle combines modified alkaline lysis method. Collected cells are re-suspended in Resuspension Buffer. Following the addition of Lysis Buffer and Neutralization Buffer to the lysate, the chromosomal DNA and cell debris will be forms an insoluble aggregate. The insoluble protein aggregate is removed following centrifugation and transfer the clear lysate to the DNA binding filter tube. The cleared lysate contains a chaotropic salt originating from Neutralization Buffer which helps the binding of the plasmid DNA on the membrane surface. The DNA binding filter which is packed with silica based membrane has enough surface area to bind up to 20㎕ of plasmid DNA. Any salts and precipitates are eliminated by addition of the Washing Buffer. Finally, highly purified plasmid DNA can be eluted with Elution Buffer or Nuclease free autoclaved distilled water.
Gel Purification
Gel Purification is designed for purification of up to 10㎕ of fragment DNA from low-melting, TAE and TBE agarose gel within 15minutes. The size range for effective purification is about 70bp~10kb. The average recovery yield exceeds 70%~90%. Elution volume can be as little as 30㎕ when concentrated product is needed. The principle of this kit is based on adsorption of DNA onto the silica based membrane by chaotropic salt. And chaotropic salts enhance melting of agarose gel but binding of DNA onto the silica based membrane that is packed in a binding column tube. Adsorption of DNA is so selective that molten agarose and salts are not adsorbed and pass through the binding column tube. Washing eliminates salts and residual agarose gel. High-purity DNA fragments are eluted with provided Elution Buffer or distilled water.
Electrophoresis
Dissolve the proper amount of agarose in 1X TAE buffer and boil, then let cool to about 60℃. Add EtBr. After carefully mixing the EtBr, slowly pour the agarose solution into the assembled gel caster. Pour slowly to avoid the formation of bubbles. Let the gel solidify at room temperature. After the gel has solidified, remove each comb carefully. If the comb seems to be stuck, pour a little D.W then remove. Check that the gel in the caster does not slide, and that the gel is fully submerged in the buffer after being fitted in the Agaro Tank. Place the Agaro Tank Lid over the Agaro Tank. Make sure that the (-) side faces up. Load the prepared samples using a multi-channel pipette onto the loading adapter. The samples will be accurately loaded onto each well. Also, check the number of the holes to reduce loading errors. Select the voltage and proceed with electrophoresis.

Parts

<groupparts>iGEM010 CBNU-Korea</groupparts>