Part:BBa_J06702:Experience

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UNIQ5d80b38bf8d23c8d-partinfo-00000000-QINU Group: ZJU-China 2015
Author: ZJU-China 2015
Preface:
There is little message about the characterrization and features of mCherry in old pages. In addition, the quality of parts varied from year to year. With an extra promoter in it, the sequnce of mCherry in 2015 (kit plate 3, well 15B) is incrrect according to the sequncing result. And mCherry in 2012 is in a "pSB1A2" backbone, which is of 4k size and doesn't correspond with message of pSB1A2 in Part Registry. So we amplified the correct gene, assembled it with standardize pSB1C3 backbone, confirmed the sequnce by enzyme digestion and sequncing and submitted the part BBa_K1668011


Summary:
1.sequncing of parts in previous year.
2.correct the part sequnce and resemble it in a standard pSB1C3 backboneBBa_K1668011

Documentation

BACKGROUND

Figure 1 Iformation of mCherry(BBa_J06702) in Part Registry.

Figure 2 Sequencing results of 2015 mCherry in 2015 kit plate 3, well 15B.

Figure 3 Double enzyme digestion of the 2012mCherry <i>(BBa_J06702) in 2012 kit plate 2, well 8E.

We used <i> mCherry BBa_J06702 as a reporter, which is one registry star and therefore comparably more reliable. According to the message in the Registry, there is no promoter in the part therefore the reporter should not be expressed(figure 1).

Figure 4 Sequencing results of 2012 mCherry<i> in 2012 kit plate 2, well 8E.

But when we transformed the <i>mCherry gene into E.coli DH5α, the E.coli turned out to become red. With the colorless control, we concluded that the mCherry was expressed and sequnced the parts. According to the results of the sequncing, the part has a promoter with it (figure 2).

Then we got the same part from kit plate in 2013 and 2012. At last, we used mCherry of 2012 in backbone pSB1A2, which is a 2k backbone. But after we have cut the backbone with XbaI and SpeI, we found that the backbone was as big as 4k (figure 3). So we had to sequnce the mCherry of 2012, which turned out to be correct (figure 4). And the backbone had an anti-ampicillin gene with it.

Therefore we concluded that the 2012 mCherry is a correct part with the wrong backbone. Now we had assembled the correct mCherry with pSB1C3 and the mCherry functions well in our other devices (device tcdA1, device plu1537 and device plu0840)

RESULTS

PLASMID CONSTRUCTION

Figure 5 Double enzyme digestion of the improvedmCherry .

5-μl samples of the double enzyme digestion products for improved mCherry BBa_K1668011 were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters.
Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis.
The DNA size standards were 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A).
Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.
Digested plasmid backbone and mCherry fragment are indicated.

It can be clearly seen that the 900bp mCherry is in the right position in agarose gel.

DNA SEQUENCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1k part shows 100% agreement with the desired sequence.

EXPRESSION

Figure 6 Tandem expression of toxin protein and mCherry shown in pipet tube.

1ml bacterium solution was added in each pipet tube and centrifuged in a speed of 12000 rpm of for 2min.

We serial express our three toxins (TcdA1, Plu1537 and Plu0840) with MCherry, shown in figure 6.
The redness of all three devices indicates that mCherry functions well.






UNIQ5d80b38bf8d23c8d-partinfo-00000001-QINU