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È¿°úÀûÀÎ ºÐºñ¹ßÇö ½Ã½ºÅÛ ½ºÅ©¸®´× ¹× ±¸ÃàÀ» À§ÇÑ

B. subtilis Secretory Protein Expression System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
3380
B. subtilis Secretory Protein Expression System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç° 
10 ȸ
776,800¿ø 
971,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3380_e.v2110Da.pdf




*º» Á¦Ç°±ºÀ» »ó¾÷Àû ¿ëµµ·Î ±¸¸ÅÇÏ´Â °æ¿ì ´ÙÄ«¶óÄÚ¸®¾Æ °í°´Áö¿ø(02-2081-2510)À¸·Î º°µµ ¹®Àǹٶø´Ï´Ù.
Á¦Ç°¼³¸í
Bacillus subtilis¸¦ ¼÷ÁÖ·Î ÇÏ´Â ÀçÁ¶ÇÕ ´Ü¹éÁúÀÇ »ý»êÀº °¡¿ëÈ­ ¹ßÇö ¹× ºÐºñ ¹ßÇö¿¡ ÀûÇÕÇϸç, ƯÈ÷ S-S°áÇÕ°ú °°ÀÌ º¹ÀâÇÑ ±¸Á¶¸¦ °¡Áö´Â ´Ü¹éÁúÀÇ »ý»ê¿¡ È¿°úÀûÀÌ´Ù. ¶ÇÇÑ, E. coli¸¦ ¼÷ÁÖ·Î ÇÏ´Â ¹ßÇö°è¿Í´Â ´Þ¸® ¿£µµÅå½Å (endotoxin)ÀÌ »ý»ê ´Ü¹éÁú¿¡ È¥ÀÔµÇÁö ¾Ê´Â ÀåÁ¡µµ ÀÖ´Ù. ¹Ý¸é¿¡ ÀÌÁ¾ ´Ü¹éÁúÀÇ ºÐºñ È¿À²Àº ½Ã±×³Î ÆéƼµå ŸÀÔ¿¡ Å©°Ô ¿µÇâÀ» Áشٴ °ÍÀÌ T. Eggert ±×·ì¿¡ ÀÇÇØ º¸°íµÇ°í ÀÖ´Ù.
ÀÌ¿¡ ´ÙÄ«¶ó´Â B. subtilisÀÇ È¿À²ÀûÀÎ ºÐºñ ¹ßÇö°è¸¦ ½ºÅ©¸®´×/±¸ÃàÇϱâ À§ÇÑ ½Ã½ºÅÛÀ» °³¹ßÇÏ¿´´Ù. º» ½Ã½ºÅÛÀ» ÀÌ¿ëÇϸé B. subtilis¿¡¼­ À¯·¡ÇÑ 173Á¾·ùÀÇ ºÐºñ ½Ã±×³Î ÆéƼµå¿¡¼­ ¸ñÀû ´Ü¹éÁúÀÇ ºÐºñ ¹ßÇö¿¡ ÀûÇÕÇÑ ½Ã±×³Î ÆéƼµå¸¦ ½ºÅ©¸®´× ÇÒ ¼ö ÀÖ´Ù. ±âÁ¸ÀÇ B. subtilis plasmid´Â copy ¼ö°¡ ¸Å¿ì Àû¾î B. subtilis¿¡¼­ plasmid ±¸ÃàÀÌ ¾î·Á¿î ´ÜÁ¡ÀÌ ÀÖ¾úÁö¸¸, º» ½Ã½ºÅÛÀÇ pBE-S DNA´Â E. coli¿Í B. subtilisÀÇ shuttle vector·Î E. coli¿¡¼­ plasmid¸¦ ±¸ÃàÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°Àº B. subtilis ºÐºñ ½Ã±×³Î ÆéƼµå DNA libraryÀÎ SP DNA mixture, B. subtilis - E. coli shuttle vectorÀÎ pBE-S DNA ¹× ´Ü¹éÁú ¹ßÇö¿ë ¼÷ÁÖ B. subtilis RIK1285°¡ Æ÷ÇԵǾî ÀÖ´Ù. SP DNA mixture´Â 173Á¾·ùÀÇ B. subtilisÀ¯·¡ ºÐºñ ½Ã±×³Î ÆéƼµå¸¦ ÄÚµùÇÏ´Â DNA´ÜÆí (In-Fusion Cloning¿ë)ÀÇ È¥ÇÕ¹°·Î ¸ñÀûÀ¯ÀüÀÚ¸¦ »ðÀÔÇÑ ÈÄ, ´ç»çÀÇ In-Fusion Cloning ±â¼úÀ» Àû¿ëÇÏ¿© ¼Õ½±°Ô ºÐºñ ½Ã±×³Î ÆéŸÀ̵带 Ãß°¡, ÃÖÁ¾ library¸¦ ±¸ÃàÇÒ ¼ö ÀÖ´Ù. pBE-S DNA¿¡´Â B. subtilis¿¡¼­ ÀÛµ¿ÇÏ´Â pUB110À¯·¡ÀÇ º¹Á¦ori (pUB ori)¿Í kanamycin ³»¼ºÀ¯ÀüÀÚ (Kanr), E. coli¿¡¼­ ÀÛµ¿ÇÏ´Â pUCÀ¯·¡ÀÇ º¹Á¦ori (ColE1 ori)°ú ampicillin³»¼ºÀ¯ÀüÀÚ (Ampr)°¡ Æ÷ÇԵǾî ÀÖ´Ù. ¶ÇÇÑ B. subtilis À¯·¡ÀÇ subtilisin promoter (aprE promoter)¿Í ºÐºñ ½Ã±×³Î ÆéƼµå (aprE SP)¸¦ Æ÷ÇÔÇÏ°í, ±× ÇÏ·ù¿¡ multicloning site (MCS) ¹× His tag ¼­¿­ÀÌ ¹èÄ¡µÇ¾î ÀÖ´Ù.
MCS¿¡ ¸ñÀûÀ¯ÀüÀÚ¸¦ »ðÀÔÇÑ pBE-S DNA¸¦ Á¦ÇÑÈ¿¼Ò Mlu I¿Í Eco52 I·Î Àý´ÜÇÏ¿© ¼±ÇüÈ­ÇÑ ÈÄ, In-Fusion Cloning systemÀ¸·Î aprE SP ´ë½Å SP DNA mixture¿¡ Æ÷ÇÔµÈ 173Á¾ÀÇ ºÐºñ ½Ã±×³Î ÆéƼµå DNA library¸¦ µµÀÔÇÏ¿© (±×¸² ÂüÁ¶), ¸ñÀû ´Ü¹éÁú¿¡ ÀûÇÕÇÑ °íºÐºñ ¹ßÇö°è¸¦ ½ºÅ©¸®´×ÀÌ °¡´ÉÇÏ´Ù. MCS´Â E. coli Cold shock expression¿ë pCold¢â vector (Code 3360, 3365, 3371 ¿Ü)¿Í °°Àº ¼­¿­À̹ǷΠ½±°Ô ¸ñÀûÀ¯ÀüÀÚ¸¦ »ðÀÔÇÒ ¼ö ÀÖ´Ù.
º» Á¦Ç°¿¡ Æ÷ÇÔµÈ ¼÷ÁÖ B. subtilis RIK1285´Â 2Á¾·ùÀÇ protease »ý»ê´É·ÂÀÌ ¾ø±â ¶§¹®¿¡, ¸ñÀû ´Ü¹éÁúÀÇ ºÐºñ ¹ßÇö¿¡ ÀûÇÕÇÏ´Ù.
±¸¼º (Library 10ȸ Á¦ÀÛºÐ)

1.

SP DNA mixture (0.032 pmol/§¡) *1

45 §¡

2.

pBE-S DNA (0.5 ¥ìg/§¡) *2

20 §¡

3.

B. subtilis RIK1285 (glycerol stock) *3

100 §¡¡¿2

*1 In - Fusion cloningÀ» À§ÇÑ 173 Á¾ B. subtilis ºÐºñ ½Ã±×³Î ÆéŸÀ̵å DNA
*2 Çü»ó: TE buffer (pH8.0)
*3 Marburg 168 derivative: trpC2, lys1, aprE ¥Ä3, nprR2, nprE18

º¸Á¸
- 80 ¡É
(SP DNA mixture ¹× pBE-S DNA´Â -20¡É¿¡¼­ º¸Á¸ °¡´É)
pBE-S DNA vector map

Sequence Á¤º¸
- pBE-S Vector
- SP DNA Mixture
»ç¿ë»óÀÇ ÁÖÀÇ
  • º» Á¦Ç°Àº In-Fusion Cloning Kit¿Í ÇÔ²² »ç¿ëÇϽʽÿÀ. ¶ÇÇÑ, °íÈ¿À² ÇüÁú Àüȯü¸¦ ¾ò±â À§ÇÏ¿©, E. coli HST08 Premium Competent CellÀ» »ç¿ëÇÏ´Â °ÍÀ» ÃßõÇÕ´Ï´Ù.
  • SP DNA mixture´Â PCR ÁõÆøÇÑ DNA fragment¸¦ È¥ÇÕÇÏ¿© Á¶Á¦ÇÏ°í ÀÖ½À´Ï´Ù. µû¶ó¼­ ºóµµ´Â ³·Áö¸¸ primer dimer µîÀÇ PCR ÁõÆø ºÎ»ê¹°ÀÌ Å¬·Î´×µÇ´Â °æ¿ì°¡ ÀÖ½À´Ï´Ù.
  • º» Á¦Ç°Àº ¿¬±¸ ¸ñÀûÀ¸·Î¸¸ »ç¿ë °¡´ÉÇÑ Á¦Ç°ÀÔ´Ï´Ù (Research use only).
    º» Á¦Ç° ¶Ç´Â º» Á¦Ç°À¸·Î ¸¸µé¾îÁø »ê¹°À» »ó¾÷Àû ¸ñÀûÀ¸·Î ÀÌ¿ëÇÏ´Â °æ¿ì, ¶óÀ̼±½º°¡ Æ÷ÇÔµÈ Á¦Ç°À» ±¸ÀÔÇÏ½Ç ¼ö ÀÖÀ¸´Ï ´ÙÄ«¶óÄÚ¸®¾Æ (02-2081-2510)·Î º°µµ ¹®Àǹٶø´Ï´Ù.
°ü·ÃÁ¦Ç°
- PCR cloning kit : In-Fusion¢ç Snap Assembly Master Mix
- °íÈ¿À² Competent Cell : E. coli HST08 Premium Competent Cell
- Â÷¼¼´ë Membrane ±â¼úÀÇ His-tagged protein Á¤Á¦ column : Capturem¢â His-Tagged Purification
- Acetic acid ¾ø´Â °í°¨µµ ´Ü¹éÁú ¿°»ö½Ã¾à : TaKaRa CBB Protein Safe Stain
- ¹ü¿ëÀûÀÎ °ËÃâºÎÅÍ ÃÊ°í°¨µµ °ËÃâ±îÁö ´Ù¾çÇÑ Á¦Ç° ¶óÀξ÷ : HRP Substrate ½Ã¸®Áî

Keyword :

Brevibacillus£¯Bacillus ¹ßÇö°è
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È¿°úÀûÀÎ ºÐºñ¹ßÇö ½Ã½ºÅÛ ½ºÅ©¸®´× ¹× ±¸ÃàÀ» ..
B. subtilis Secretory Protein Expression System