Team:NTU Taida/Circuit2

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Illustrations can help you understand:</p>
Illustrations can help you understand:</p>
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<h5 style="line-height:1.4em;font-size:110%;"><b>1.PelB (66mer)</b></p>
<h5 style="line-height:1.4em;font-size:110%;"><b>1.PelB (66mer)</b></p>
GAATTC GCGGCCGC T T<em style="background-color:yellow">CTAG ATG AAA TAT CTG CTG CCG ACC GCG GCG</em><em style="background-color:LightGreen"> GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG T A</em>CTAGT A GCGGCCG CTGCAG</p>
GAATTC GCGGCCGC T T<em style="background-color:yellow">CTAG ATG AAA TAT CTG CTG CCG ACC GCG GCG</em><em style="background-color:LightGreen"> GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG T A</em>CTAGT A GCGGCCG CTGCAG</p>

Latest revision as of 01:49, 18 October 2014

NTU-Taida

Skin Whitening ---PKEK

  • Inspiration :

    Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. One of the most often used skin lighteners is hydroquinone or the hydroquinone glycoside of arbutin. However, these compounds have a cytotoxic effect on melanocytes and have an irritating effect on the skin. There is thus an increasing need for improved active ingredients for the treatment of hyperpigmentation, but also for the purely cosmetic lightening of relatively large areas of pigmented skin that are entirely appropriate to the individual skin.

    The tetrapeptide PKEK (Pro-Lys-Glu-Lys) is described in the patent application WO/2008/085494 and in WO/2009/068351. It has immunomodulatory properties and also anti-aging efficacy .

    According to researches, PKEK may be delivered upon topical application into human skin where they subsequently exert biological effects on skin cells, including epidermal keratinocytes and dermal fibroblasts effects .
  • Synthetic Peptides Sequence :

    Here are three kinds of signal sequence:
    1.PelB (66mer) ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG

    2.OmpA (63mer) ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCG CAG GCG

    3.PhoA (63mer) ATG AAA CAG AGC ACC ATT GCG CTG GCG CTG CTG CCG CTG CTG TTT ACC CCG GTG ACC AAA GCG

    Unlike others, we have our own way to synthesize the signal sequence.

    It’s common to order whole gene sequences to Biotech, but the cost is always shocking, and since the budget limitation, we strive to find our way out.

    Here is our strategy:

    First, we ordered 4 primers which can combine to each other, and put them into the plasmid backbone.

    Second, we link the iGEM’s official prefix and suffix (EcoRI, XbaI, SpeI and PstI) to our signal sequence, and choose XbaI、SpeI as restriction sites.

    Illustrations can help you understand:

    1.PelB (66mer)

    GAATTC GCGGCCGC T TCTAG ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG T ACTAGT A GCGGCCG CTGCAG

    CTTAAG CGCCGGCG A AGATC TAC TTT ATA GAC GAC GGC TGG CGC CGC CGC CGC GAC GAC GAC GAC CGC CGC GTC GGC CGC TAC CGC A TGATCA T CGCCGGC GACGTC

    2.OmpA (63mer)

    GAATTC GCGGCCGC T TCTAG ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCG CAG GCG T ACTAGT A GCGGCCG CTGCAG

    CTTAAG CGCCGGCG A AGATC TAC TTT TTT TGG CGC TAA CGC TAA CGC CAC CGC GAC CGC CCG AAA CGC TGG CAC CGC GTC CGC A TGATCA T CGCCGGC GACGTC

    3.PhoA (63mer) GAATTC GCGGCCGC T TCTAG ATG AAA CAG AGC ACC ATT GCG CTG GCG CTG CTG CCG CTG CTG TTT ACC CCG GTG ACC AAA GCG T ACTAGT A GCGGCCG CTGCAG

    CTTAAG CGCCGGCG A AGATC TAC TTT GTC TCG TGG TAA CGC GAC CGC GAC GAC GGC GAC GAC AAA TGG GGC CAC TGG TTT CGC A TGATCA T CGCCGGC GACGTC

    The yellow, green, blue and red part in pictures are primers we order from Biotech. The combination of these four sequences will produce the sticky ends of XbaI and SpeI. We ligase the yellow and blue part with green and red one respectively. Finally, we linked these two parts with plasmid backbones treated with Xbal and Spel.

    Moreover, we should notice the possibility of antisense considering the sticky ends of XbaI and SpeI will pair together. After all the ligation and transformation process, we should use colony PCR to pick up out correct colony. On PSB1C3, there are two sequence: V2F and VR, provide the primer binding site. Therefore, while choose V2F and VR to be primers, we could still see band on gel even there’s no insertion. Our experiment design can intelligently solve this problem. We use one of our signal sequences to replace VR sequence, so the cases of antisense and without insert will not have result during electrophoresis.

    All signal sequences are cloned and ligated on pSB1C3 plasmid backbone, which can be applied broadly as biobricks. By doing so, we significantly lower the cost compared to directly synthesize the nucleic acid sequences. And it is suitable for shorter gene sequence. We think it worth referencing by other iGEM teams.

    PKEK sequence

    In gram-negative bacteria, different types of protein secretion systems have been described , and we decided to put PKEK sequence directly behind the signal sequence, because it has only 18 nucleic acids. PKEK (18mer)

    CGC AAA GAA AAA TAA TAA

  • Background Knowledge :

    Regulation of skin pigmentation is a highly comp process that in addition to melanin-synthesizing melanocytes involves the production of pigmentation-inducing soluble factors by epidermal keratinocytes including proinflammatory cytokines such as IL-6, IL-8 as well as alpha-melanocyte-stimulating hormone (a-MSH). And previously shown that as a general principle specific tetrapeptides may be delivered upon topical application into human skin where they subsequently exert biological effects on skin cells, including epidermal keratinocytes and dermal fibroblasts, and significantly modulating skin pigmentation . Furthermore, PKEK was equally capable of reducing POMC in UV-irradiated keratinocytes and in skin. Thus, PKEK interferes with the keratinocyte to melanocyte signaling processes involved in skin pigmentation was shown to reduce tyrosinase expression in vivo .
  • Reference :

    Modulation of skin pigmentation by the tetrapeptide PKEK: in vitro and in vivo evidence for skin whitening effects DOI:10.1111/j.1600-0625.2011.01415.x

    fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5974-2
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