Regulatory
Gal4-UAS

Part:BBa_K1884004

Designed by: Chengrong Xie   Group: iGEM16_SZU-China   (2016-10-07)
Revision as of 23:02, 19 October 2016 by Nllenwalker (Talk | contribs) (→‎Biology)


Upstream activating sequence

An upstream activating sequence(UAS) is a cis-acting regulatory sequence which is a region of non-coding DNA, regulating the transcription of nearby genes.

Biology

Due to this part's essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the enhancer in multicellular eukaryotes.[1] Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity.[2] The upstream activation sequence is found adjacently upstream to a minimal promoter (TATA box) and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin.[3] To further understand the function of an upstream activation sequence, it is beneficial to see its role in the cascade of events that lead to transcription activation. The pathway begins when activators bind to their target at the UAS recruiting a mediator. A TATA-binding protein subunit of a transcription factor then binds to the TATA box, recruiting additional transcription factors. The mediator then recruits RNA polymerase II to the pre-initiation complex. Once initiated, RNA polymerase II is released from the complex and transcription begins.[4]

Usage

In our light-mediate controlled yeast-two-hybrid system, the function of UAS is to regulate the transcription.when CRY2 is disconnected with CIB1, the transcription of entire system will halt at UAS until the CIB1 and CRY2 conbined with each other under blue light, the genes which are on the 3 primer end of UAS will be transcripted.Fig 1

Figure 1. The diagram of light-mediate controlled yeast-two-hybrid system.


To make this parts RFC 10 competible, we use Site-directed mutagenesis to mutate the old sequence so that none of these four Restriction enzyme(EcoR1/Xba1/Sep1/Pst1) can digest this gene. (Fig 2.)

Figure 2. the location of the mutated basic group.


UAS is 179bp in length. Fig.3 shows the DNA sequence of UAS is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of UAS PCR product is rather high compared with DNA Marker, which indicates that the PCR product of UAS is in a high concerntration.

Figure 3. The electrophoretogram of UAS PCR product.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 109
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 38
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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