Difference between revisions of "Part:BBa J06702:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
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===User Reviews=== | ===User Reviews=== | ||
<!-- DON'T DELETE --><partinfo>BBa_J06702 StartReviews</partinfo> | <!-- DON'T DELETE --><partinfo>BBa_J06702 StartReviews</partinfo> | ||
− | < | + | '''Group''': ZJU-China 2015 |
− | + | <br> | |
− | + | '''Author''': ZJU-China 2015 | |
− | + | <br> | |
− | <partinfo> | + | '''Preface''': |
− | < | + | <br> |
− | + | <br> | |
− | + | '''Summary''': | |
− | | | + | <br> |
+ | <br> | ||
+ | <br> | ||
+ | <h1>Documentation</h1> | ||
+ | he <i>mCherry</i>is an improved part from BBa_J06702 in Part Registry, composed of RBS [https://parts.igem.org/Part: BBa_B0034], reporter <i>mCherry</i> [https://parts.igem.org/Part: BBa_J06702] and double terminator [https://parts.igem.org/Part: B0010] [https://parts.igem.org/Part: B0012] | ||
+ | <br> | ||
+ | <br> | ||
+ | We found out the sequence of<i> mCherry</i> in 2015 kit plate 3(well 15B) has a promoter with it, therefore doesn’t match the information in Part Registry. Then we sequenced <i>mCherry</i> in 2013, which proved to be correct but in a pSB1A2 backbone, and standardized the correct sequence by assemble the part with standard pSB1C3 backbone. | ||
+ | <br> | ||
+ | <br> | ||
+ | <!-- --> | ||
+ | <span class='h3bb'>Sequence and Features</span> | ||
+ | <partinfo>BBa_K1668011 SequenceAndFeatures</partinfo> | ||
+ | |||
+ | |||
+ | <!-- Uncomment this to enable Functional Parameter display | ||
+ | ===Functional Parameters=== | ||
+ | <partinfo>BBa_K1668011 parameters</partinfo> | ||
+ | <!-- --> | ||
+ | |||
+ | <h2>'''Characterization'''</h2> | ||
+ | |||
+ | <h3> BACKGROUND </h3> | ||
+ | [[File:ZJU-CHINA_mCherry_figure1.png|200px|thumb|left|Figure 1 Iformation of <i> mCherry</i>(BBa_J06702) in Part Registry.]] | ||
+ | [[File:ZJU-CHINA_2015_mCherry.png|200px|thumb|left|Figure 2 Sequencing results of 2015 <i>mCherry</i> in 2015 kit plate 3, well 15B.]] | ||
+ | [[File:ZJU-CHINA_mCherry_figure3.png|200px|thumb|right|Figure 3 Double enzyme digestion of the 2012<i>mCherry <i>(BBa_J06702) in 2012 kit plate 2, well 8E.]] | ||
+ | <br> | ||
+ | We used <i> mCherry</i> [https://parts.igem.org/Part: 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). | ||
+ | <br> | ||
+ | <br> | ||
+ | [[File:ZJU-CHINA_2015_mCherry.png|200px|thumb|left|Figure 4 Sequencing results of 2012 <i>mCherry<i> in 2012 kit plate 2, well 8E.]] | ||
+ | But when we transformed the <i>mCherry</i> gene into <i>E.coli DH5α</i>, the <i>E.coli</i> turned out to become red. With the colorless control, we concluded that the <i>mCherry</i> was expressed and sequnced the parts. According to the results of the sequncing, the part has a promoter with it (figure 2). | ||
+ | <br> | ||
+ | <br> | ||
+ | Then we got the same part from kit plate in 2013 and 2012. At last, we used <i>mCherry</i> 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 <i>mCherry</i> of 2012, which turned out to be correct (figure 4). And the backbone had an anti-ampicillin gene with it. | ||
+ | <br> | ||
+ | <br> | ||
+ | Therefore we concluded that the 2012 <i>mCherry</i> is a correct part with the wrong backbone. Now we had assembled the correct <i>mCherry</i> with pSB1C3 and the<i> mCherry</i> functions well in our other devices (device tcdA1, device plu1537 and device plu0840) | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <h3> RESULTS </h3> | ||
+ | <h4> PLASMID CONSTRUCTION </h4> | ||
+ | [[File:ZJU-CHINA_mCherry_figure5.png|200px|thumb|left|Figure 5 Double enzyme digestion of the improved<i>mCherry </i>.]] | ||
+ | <br> | ||
+ | 5-μl samples of the double enzyme digestion products for improved <i>mCherry</i> BBa_K1668011 were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. | ||
+ | <br> | ||
+ | Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis. | ||
+ | <br> | ||
+ | The DNA size standards were 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A). | ||
+ | <br> | ||
+ | Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. | ||
+ | <br> | ||
+ | Digested plasmid backbone and <i>mCherry</i> fragment are indicated. | ||
+ | <br> | ||
+ | <br> | ||
+ | It can be clearly seen that the 900bp <i>mCherry</i> is in the right position in agarose gel. | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <h4> DNA SEQUENCING </h4> | ||
+ | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1k part shows 100% agreement with the desired sequence. | ||
+ | <br> | ||
+ | <br> | ||
+ | <h4> EXPRESSION </h4> | ||
+ | [[File:ZJU-CHINA_expression.png|200px|thumb|left|Figure 6 Tandem expression of toxin protein and <i> mCherry </i>shown in pipet tube. ]] | ||
+ | <br> | ||
+ | <br> | ||
+ | 1ml bacterium solution was added in each pipet tube and centrifuged in a speed of 12000 rpm of for 2min. | ||
+ | <br> | ||
+ | <br> | ||
+ | We serial express our three toxins (TcdA1, Plu1537 and Plu0840) with MCherry, shown in figure 6. | ||
+ | <br> | ||
+ | The redness of all three devices indicates that <i>mCherry</i> functions well. | ||
+ | |||
+ | <br> | ||
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Revision as of 15:38, 16 September 2015
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_J06702
User Reviews
UNIQe0b9c632f28a7799-partinfo-00000000-QINU
Group: ZJU-China 2015
Author: ZJU-China 2015
Preface:
Summary:
Documentation
he mCherryis an improved part from BBa_J06702 in Part Registry, composed of RBS BBa_B0034, reporter mCherry BBa_J06702 and double terminator B0010 B0012
We found out the sequence of mCherry in 2015 kit plate 3(well 15B) has a promoter with it, therefore doesn’t match the information in Part Registry. Then we sequenced mCherry in 2013, which proved to be correct but in a pSB1A2 backbone, and standardized the correct sequence by assemble the part with standard pSB1C3 backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
BACKGROUND
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).
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
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
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.
UNIQe0b9c632f28a7799-partinfo-00000002-QINU