Team:Gifu/Results

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Results

PCR

The result of PCR is shown in Fig.1. All PCR was performed by primers in table 1. All PCR were succeeded. We used PrimeSTAR Max premix for amplification.


Fig.1 PCR of BBa_K1491033

Fig.1 PCR of Seq.1

Fig.1 PCR of Seq.2

Cell-free expression

Here we show the result of expression. (Fig. 4~7) Seq.2 (monomer RFP, mRFP) was expressed well in PURE system and slightly expressed in myTXTL. Seq.1 (polymer RFP, pRFP) has no fluorescence.


Fig.4 Expression in PURE system (No UV)

P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA

mRFP tem. and mRFP PCR was slightly turned into red.


Fig.4 Expression in PURE system (No UV)

P: Positive (eGFP), N: Negative (Water)

Positive (eGFP) shows green color.


Fig.4 Expression in PURE system under UV

P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA

mRFP shows the clear fluorescence. mRFP tem. and mRFP PCR shows the same result. mRFP shown in the figure is mRFP tem.


Fig.4 Expression in mtTXTL under UV

P: Positive (eGFP), N: Negative (Water)

Positive (eGFP) turned into green.

SDSPAGE

We performed SDSPAGE to confirm whether pRFP was expressed or not. The result is shown below.

Fig.4 SDSPAGE

As a result of SDS-PAGE, both myTXTL and PURE system shows the intense expression of the protein. In PURE system, we could see the monomer RFP. With the eye, a slight expression of mRFP in myTXTL was confirmed but not in SDSPAGE. In both cell-free system, the long-chain protein and mRFP was not confirmed with Seq.1.

Discussion

3-dimensional structure of the enhancer, stem loop structure may inhibit the function of ribozymes. Ribozymes need to maintain certain structures to catalyze the reaction. Chemically, it is possible that the enhancer changes the ribozymes' conformation. We need to make the same plasmid without the enhancer, so that we can confirm whether the stem loop structure effects on the ribozymes.

Furthermore, in myTXTL, even we confirmed the large expression of eGFP, we could not see enough expression of RFP. This may be because our T7 RNA polymerase does not fit in myTXTL crude extract. We should change it into T7 RNA polymerase from Thermo at a conc. of 2 U/μL. This is functional and confirmed by the company.

This time we did not use TEV protease site. Before performing the cut, we should consider whether no indispensable components for transcription and translation do not have the site. In addition, TEV protease can work in low temperature. To make monomer RFP, incubation temperature can be at low temperature. With this tip, folding rate of the expressed protein would be much slower and the cutting rate may be effective.