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<p>Therefore we used the standard 3A-Assembly protocol. The constitutive promoter (BBa_J23102) was provided in pSB1A3, our constructed Biobrick (BBa_K1860700) is carried in pSB1C3, hence the assembly was performed on pSB1K3.
 
<p>Therefore we used the standard 3A-Assembly protocol. The constitutive promoter (BBa_J23102) was provided in pSB1A3, our constructed Biobrick (BBa_K1860700) is carried in pSB1C3, hence the assembly was performed on pSB1K3.
  
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Revision as of 18:42, 18 September 2015

Project Results

miRNA2911 (BBa_K1860700)

Aim

Our goal was to use synthetic biology to produce miRNA2911, which is endogenously expressed in Lonicera japonica.

Results

We were able to constructed a Biobrick (BBa_K1860700) that contains the anti-viral miRNA2911. The Biobrick was validated by sequencing.

Design of Oligos

We started our project by designing oligos of the miRNA2911. It was really important to consider that the desired sequence had to be in line with the iGEM standard. Meaning that the resulted Biobrick can be used for 3A-Assemblies later on. Hence the miRNA2911 is flanked by the appropriate restriction sites (EcoRI, XbaI, SpeI, PstI). Depicted in Figure 1 are the designed sequences.

Annealing of Oligos

In the next step the oligos needed to be annealed. We used the standard ‘Annealing Protocol’ and Figure 2 depicts the results of this annealing. The appropriate bands of the resulted miRNA2911 insert are at 61 bp.

Ligation

After confirmation of successful annealing we proceeded with the ligation of the insert (miRNA2911) into the standard backbone pSB1C3. For this purpose the linearized backbone (pSB1C3) was cut with EcoRI and PstI and the insert was ligated into the vektor. The insert must not be cut as it was designed to form overhangs after annealing.

Shown in the following Figure is the verification of ligation. The ligation was verified positive as the ligated vector runs at 2050 bp and the control sample, only containing pSB1C3 runs at around 2000 bp, as seen in the agarose gel.

Following, the ligated product was transformed into the E.coli strain DH5α. Single colonies were picked, liquid culture were grown overnight and plasmids were prepped.

Verification

Colony PCR

To be able to verify if our E.coli actually carry our desired plasmid a colony PCR was performed. Therefore an aliquot of the liquid culture was again plated out and grown overnight. Sixteen different colonies were tested using the standard sequencing primers. The results of six of these PCRs are shown in the following Figure. The gel verifies that the E.coli are carrying our desired vector containing the miRNA2911.

Sequencing

Finally we verified our new Biobrick by sequencing.

constitutive promoter and miRNA (BBa_K1860702)

We combined the generated BioBrick (BBa_K1860700), containing the miRNA2911, with a constitutive promoter (BBa_J23102) that was provided with the Standard Distribution by 3A-Assembly. This is necessary to further on be able to test if miRNA2911 is:

  • actually expressed
  • stable in E.coli and
  • how it is actually folded

    • Therefore we used the standard 3A-Assembly protocol. The constitutive promoter (BBa_J23102) was provided in pSB1A3, our constructed Biobrick (BBa_K1860700) is carried in pSB1C3, hence the assembly was performed on pSB1K3.

    Fig. 2: Our BioBrick as an important intermediate step

    GroEL - heat-inducible promoter (BBa_K1860701)

    Design of Oligos

    GroEl is a heat-inducible promoter and we started by designing oligos of GroEL. The sequence was kindly provided by Prof. Ignatova. Just like for the design of the miRNA2911 here we also needed to reconsider the that the desired sequence containing GroEL had to be in line with the iGEM standard. Hence also the GroEL promoter is flanked by the appropriate restriction sites (EcoRI, XbaI, SpeI, PstI).

    Annealing of Oligos

    In the next step the oligos needed to be annealed. We used the standard ‘Annealing Protocol’ and the following Figure depicts the results of this annealing. The appropriate bands of the resulted GroEL insert are at 91 bp.

    Ligation

    After confirmation of successful annealing we proceeded with the ligation of the insert (GroEL) into the standard backbone pSB1C3. For this purpose the linearized backbone (pSB1C3) was cut with EcoRI and PstI and the insert was ligated into the vector. The insert must not be cut as it was designed to form overhangs after annealing.

    Due to submission deadline the BioBrick plasmid was sent to the registry without sequencing.

    pezT

    Aim

    Aim was to generate a BioBrick containing the PezT gene for autolysis as shown under “project → kill gene”. We wanted to amplifiy the gene from a plasmid provided by Prof. Meinhard (Robert Koch Institute, Heidelberg, Germany) via PCR. To get rid of illegal restriction sites as PstI we planed a mutagenesis PCR. Finally the PezT gene should get ligated into the pSB-1C3 backbone.

    Results

    We designed primer for mutagenesis and start first PCR steps but have to stop due to submission deadline.


    We thank our sponsors: