Team:SYSU-China/page project modules construction


Modules Construction-Sun Yat-sen Univ.

    We have designed three categories of modules - Expressional Testing Modules, Functionalizing Testing Modules and Final Functional Modules - to test the expression and function of every element and to accomplish the final function. The followings are the details of these modules.

1. Expression Testing Modules

    In order to test whether the elements we obtain from E.coli genome PCR can express normally, we have constructed the expressional testing modules through genetic engineering methods. But the existing promoter in the pET series plasmids will affect the expression test of promoter RecA and RecN, so we cut off the T7 promoter of the plasmid pET28a. By digesting promoter RecA and RecN with restriction endonuclease EcoRI and XbaI, digesting EGFP gene with PstI and HindIII, and then ligating them into plasmid pET28a, pET32a and pUC18 with T4 ligase, the following testing modules have been successfully constructed:

  • a) RecA-gfp-pET28a(promoter knock-out) (figure.1)
  • b) RecN-gfp-pET28a(promoter knock-out) (figure.2)
  • c) CheZ(no terminator)-gfp-pUC18 (figure.3)
  • d) TrkD(no terminator)- gfp-pUC18 (figure.4)
  • e) CheZ-pET32a (figure.5)
  • f) TrkD-pET32a (figure.6)

  •     Through the appearance of the green fluorescence with the first four modules, we can know whether the elements RecA, RecN, CheZ and TrkD express normally as we expected. With the Western-blot tests using the modules CheZ-pET32a and TrkD-pET32a, we can directly know the expression state of CheZ and TrkD.

    2. Functionalizing Testing Modules

        For the purpose of realizing the function that CheZ and TrkD can start to express under the induction of nuclear radiation, we expected to ligate RecA with CheZ, and RecN with TrkD. But the ligation of RecN and TrkD was of great difficulty to be accomplished. This may be caused by the facts that RecN is too short and the overlapping zone of RecN and TrkD has far less than 15 base pairs, which is essential for recombinant PCR. However, TrkD expression under the promotion of RecA satisfies our requirements. As a result, we have constructed the following modules to conduct the functionalizing tests:

  • a) RecA-CheZ-pET28a(promoter knock-out) (figure.7)
  • b) RecA-TrkD-pET28a(promoter knock-out) (figure.8)
  • c) RecA- CheZ(no terminator)-gfp-pET28a(promoter knock-out) (figure.9)
  • d) RecA- TrkD(no terminator)-gfp-pET28a(promoter knock-out) (figure.10)

  •     In addition to the modules constructed above, we also intend to construct the following module:

  • e) RecN-TrkD(no terminator)-ag43- pET28a(promoter knock-out)

  •     With the first two modules, we could test the expression of CheZ and TrkD through the directed motion of E.coli and the Cs+ absorption under the induction of radiation. With the next two modules, the expression of CheZ and TrkD under the radiation induction can be reflected directly through the fluorescence of gfp. With the last module, we could test the expression state of ag43 through whether E.coli cells connect with each other.

    3. Final Functional Modules

        Based on the former construction of plasmids, we expect to integrate the module RecA-CheZ and RecA-TrkD-ag43 in the same E.coli to fulfill the final function that the E.coli could swimming to the source of nuclear leakage and absorb the radioactive Cs+ under the induction of radiation. At the same time, the ag43 in the downstream of TrkD starts to express. When the ag43 protein accumulates above the threshold, E.coli cells could cross link each other, and then be collected to clear the radioactive.

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    From the 2011 iGEM team SYSU-China (2011)

    Sun Yat-Sen University, Guangzhou, China

    Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China

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