Team:UESTC Life/Results and discussion

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{| style="color:#1b2c8a;background:transparent;" cellpadding="3" cellspacing="1" border="0" bordercolor="red" width="80%" align="center"
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!align="center"|<font size="200px">Results and Discussion</font>
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        <a href="https://2013.igem.org/Team:UESTC_Life#/page/5"><img src="https://static.igem.org/mediawiki/igem.org/8/8a/Uestclifebiot_logoarrow.png" style="filter:alpha(opacity=0,finishopacity=60,style=0)"></a>
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== '''TCP Biodegradation Achieved''' ==
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:<font size=4>Growth of ''E.coli'' strain ''MC1061'' transformed ''DhaA+P2A+HheC'' and ''DhaA'' gene in LB medium with inducer and 5mM TCP respectively, as the substrate was monitored in batch culture. The concentration of the products were confirmed using GC column(AC5). Growth resulted in disappearance of the substrate and simultaneous formation of biomass, 2,3-DCP,  epichlorohydrin, chloropropanol, and glycerol. The result was screened by GC analysis. Above the degradation of TCP and the intermediate product 2,3-DCP indicated that multistep biodegradation was happened. (Fig.1 and Fig.2). When the ''E.coli'' carried ''DhaA+P2A+HheC'' gene cultivated in medium with 2,3-DCP, the concentration of epichlorohydrin have been detected(Fig.2). 2,3-DCP was degraded to epichlorohydrin. Chloropropanol and glycerol couldn’t be detected by our columns. Since the two active enzymes were in bacteria, if the degradation followed the theoretic pathway (refer to project), TCP would be degraded to glycerol in the end.  In the future, new columns and methods will be used to further proof the result.</font>
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 +
https://static.igem.org/mediawiki/igem.org/4/4a/Uestclifer1.1.png  <br/>
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Fig.1. ''E.coli'' strain ''MC1061'' carried ''DhaA+P2A+HheC'' gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP was degraded at the same time.<br/>
 +
 +
 +
https://static.igem.org/mediawiki/igem.org/4/4c/Uestclifer1.2.png<br/>
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Fig.2. ''E.coli'' strain ''MC1061'' carried ''DhaA'' gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP couldn’t be degraded.<br/>
 +
 +
https://static.igem.org/mediawiki/igem.org/5/5e/Uestclifer1.3.png<br/>
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Fig.3. ''E.coli'' strain ''MC1061'' carried ''DhaA+P2A+HheC'' gene incubated in LB medium with 2,3-DCP(5mM) at 30℃. The concentration of 2,3-DCP and its mediate product epichlorohydrin were measured by GC.<br/>
 +
 +
== '''γ-HCH Biodegradation and F2A Cleaving Achieved'''==
 +
 +
:<font size=4>Growth of ''E.coli'' strain ''MC1061'' transformed ''LinA+F2A+LinB'' and ''LinA'' gene in LB medium with inducer and 5Mm γ-HCH  respectively, as the substrate was monitored in batch culture. The result was screened by GC analysis(Fig.4). Growth resulted in disappearance of the substrate and simultaneous formation of biomass and γ-PCCH et al. But available columns were limited, hence the intermediate product couldn’t be detected. Through SDS-PAGE analysis, F2A peptide could cleave LinA and LinB(Fig.5). LinB would degrade the intermediate product of γ-HCH and according to theory, the γ-HCH could be degraded to desired low toxic compounds.</font>
 +
 +
https://static.igem.org/mediawiki/igem.org/c/c5/Uestclifer2.1.png <br/>
 +
Fig.4. ''E.coli'' strain ''MC1061'' carried ''LinA'' and ''LinA+F2A+LinB'' gene incubated in LB medium with γ-HCH(5mM) at 30℃. The concentration of γ-HCH was measured by GC. The bacteria carried gene ''LinA+F2A+LinB'' degraded γ-HCH(5mM) but the intermediate product couldn't be detected. Blank control(empty vector) have been added, the concentration of γ-HCH merely remained unchanged.
 +
 +
https://static.igem.org/mediawiki/igem.org/0/05/Uestclifenote11.jpg <br/>
 +
FIG.5. SDS-PAGE analysis. Supernatant and sediment collected after breaking cell. Lane1 and lane2 are blank controls, in the lane3 and lane5 LinA and LinB are solved in the buffer; in the lane7 and lane8, F2A peptide cleaved LinA and LinB, both of them are solved. The most of protein, LinA+F2A+LinB, is in the sediment as a kind of inclusion body.
 +
 +
=='''P2A Being An Excellent Linker in Chimeric Protein''' ==
 +
 +
:<font size=4>As for P2A peptide sequence, it didn’t cleave the DhaA and HheC, and this chimeric protein was linked by P2A peptide. (Fig.6).</font><br/>
 +
 +
:<font size=4>''DhaA+P2A+HheC'' gene were expressed in ''E.coli'' strain ''MC1061''. Separating supernatant and sediment of the broken cell liquid and used colorimetric method to detect the activity with TCP and 1,3-DCP.(Fig.7). DhaA+P2A+HheC in supernatant had activity of DhaA and HheC enzymes, while in sediment, there wasn't any activity.</font><br/>
 +
 +
:<font size=4>HheC has a strict structure. Only if four HheC subunits compose together as a tetramer can it catalyze dehalogenation. Measuring the activity of HheC in chimeric protein DhaA+P2A+HheC is an ideal way to detect the effect of P2A peptide. The purified enzymes, HheC and DhaA+P2A+HheC chimeric protein was isolated by AKTA FPLC. Using substrate 1,3-DCP, HheC specific activity was 6.72(U/mg) and the chimeric protein specific activity is 5.28(U/mg). It indicated that the 2A peptide was an excellent linker to compose the two enzymes together.</font><br/>
 +
 +
https://static.igem.org/mediawiki/igem.org/6/65/Uestclifenote12.jpg <br/>
 +
Fig.6 lane7 and lane8 are blank controls, lane1 and lane3 are the expression of ''DhaA'' and ''HheC'' gene. In the lane5 and lane6, HheC and DhaA bands couldn’t be found,there is DhaA+P2A+HheC chimeric protein band, P2A peptide sequence can’t cleave DhaA and HheC. In the sediment, it is DhaA+P2A+HheC inclusion body.<br/>
 +
 +
https://static.igem.org/mediawiki/igem.org/9/96/Uestclifer3.2.png  https://static.igem.org/mediawiki/igem.org/9/93/Uestclifer3.3.png<br/>
 +
Fig.7 the reaction in buffer(50MmTris-SO3,PH=8.0), after breaking cell, the supernatant and sediment were separated. Blank control was the cell transformed empty vector. Colorimetric method is a way of detecting the concentration of Cl^-(refer to protocol). The solved chimeric protein DhaA+P2A+HheC could degrade 1,3-DCP and TCP. The inclusion protein didn’t have any activity. <br/>
 +
 +
== '''Polycistronic Co-expression System Constructed ''' ==
 +
 +
:<font size=4>In polycistronic co-expression system, the quantity of HheC was less than DhaA and LinB is less than LinA(Fig.9). Because the further the distance between coding and promoter is, the less quantity of expression is. That was why we wanted to utilize 2A peptide in prokaryotic system.</font><br/>
 +
 +
https://static.igem.org/mediawiki/igem.org/d/d4/Uestclifer4.1.png
 +
https://static.igem.org/mediawiki/igem.org/8/85/Uestclifenote22.png  <br/>
 +
Fig.9 (a) In the lane5 and lane6, ''LinA'' and ''LinB'' are expressed, according to the bands, the quantity of LinB is less than LinA. (b) In the lane1 and lane2, there are both bands of DhaA  and  HheC, the quantity of DhaA is more than HheC. <br/>
 +
 +
== '''Vectors and Parts''' ==
 +
https://static.igem.org/mediawiki/igem.org/0/07/POHC00.jpg '''pOHC_00  empty modified pBAD vector'''
 +
<br/>
 +
https://static.igem.org/mediawiki/igem.org/e/e7/POHC01.png '''pOHC_01  ''LinA'' at pBAD vector'''<br/>
 +
https://static.igem.org/mediawiki/igem.org/c/cf/POHC02.png
 +
'''pOHC_02  ''LinB'' at pBAD vector'''<br/>
 +
https://static.igem.org/mediawiki/igem.org/8/8d/POHC03.png
 +
'''pOHC_03 '' DhaA'' at pBAD vector'''
 +
<br/>https://static.igem.org/mediawiki/igem.org/3/34/POHC04.png
 +
'''pOHC_04  ''HheC'' at pBAD vector'''
 +
<br/>https://static.igem.org/mediawiki/igem.org/3/33/POHC05.png
 +
'''pOHC_05  ''LinA+F2A+LinB'' at pBAD vector'''
 +
<br/>https://static.igem.org/mediawiki/igem.org/d/d2/POHC06.png
 +
'''pOHC_06  ''DhaA+P2A+HheC'' at pBAD vector'''
 +
<br/>https://static.igem.org/mediawiki/igem.org/3/32/POHC07.jpg
 +
'''pOHC_07  ''LinA+RBS+LinB'' at pBAD vector'''
 +
<br/>https://static.igem.org/mediawiki/igem.org/4/49/POHC08.jpg
 +
'''pOHC_08  ''DhaA+RBS+HheC'' at pBAD vector'''
 +
<br/>
 +
 +
 +
<font size=4>Parts we submitted<br/>
 +
 +
BBa_K1199041    ''LinA'' at pSBC31<br/>
 +
BBa_K1199042    ''LinB'' at pSBC31<br/>
 +
BBa_K1199043    '' DhaA31'' at pSBC31<br/>
 +
BBa_K1199044    ''HheC/W249P'' at pSBC31<br/>
 +
BBa_K1199045    ''LinA+F2A+LinB'' at pSBC31<br/>
 +
BBa_K1199046    ''DhaA31+P2A+HheC/W249P'' at pSBC31<br/>
 +
BBa_K1199047    ''LinA+RBS+LinB'' at pSBC31<br/>
 +
BBa_K1199049    ''DhaA31+RBS+HheCW249P'' at pSBC31<br/>
 +
</font>
 +
 +
=='''Future Work'''==
 +
 +
'''●  Assaying  the  change  of intermediate  products'''<br/>
 +
 
 +
 +
 +
'''●  Improve  the  activity  of  DhaA  and  HheC'''<br/>
 +
 +
 +
 +
'''●  Decreasing  inclusion  body'''<br/>
 +
 +
 +
 +
'''●  Using  P2A  to  create  other  chimeric  protein'''
 +
 +
=='''Achievement'''==
 +
https://igem.org/2013_Judging_Form?id=1199
 +
 +
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    <a href="https://2013.igem.org/Team:UESTC_Life#/page/5"><img src="https://static.igem.org/mediawiki/2013/6/68/Uestclifeback.png" /></a>
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Latest revision as of 15:13, 27 September 2013

Results and Discussion


Contents

TCP Biodegradation Achieved

Growth of E.coli strain MC1061 transformed DhaA+P2A+HheC and DhaA gene in LB medium with inducer and 5mM TCP respectively, as the substrate was monitored in batch culture. The concentration of the products were confirmed using GC column(AC5). Growth resulted in disappearance of the substrate and simultaneous formation of biomass, 2,3-DCP, epichlorohydrin, chloropropanol, and glycerol. The result was screened by GC analysis. Above the degradation of TCP and the intermediate product 2,3-DCP indicated that multistep biodegradation was happened. (Fig.1 and Fig.2). When the E.coli carried DhaA+P2A+HheC gene cultivated in medium with 2,3-DCP, the concentration of epichlorohydrin have been detected(Fig.2). 2,3-DCP was degraded to epichlorohydrin. Chloropropanol and glycerol couldn’t be detected by our columns. Since the two active enzymes were in bacteria, if the degradation followed the theoretic pathway (refer to project), TCP would be degraded to glycerol in the end. In the future, new columns and methods will be used to further proof the result.

Uestclifer1.1.png
Fig.1. E.coli strain MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP was degraded at the same time.


Uestclifer1.2.png
Fig.2. E.coli strain MC1061 carried DhaA gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP couldn’t be degraded.

Uestclifer1.3.png
Fig.3. E.coli strain MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with 2,3-DCP(5mM) at 30℃. The concentration of 2,3-DCP and its mediate product epichlorohydrin were measured by GC.

γ-HCH Biodegradation and F2A Cleaving Achieved

Growth of E.coli strain MC1061 transformed LinA+F2A+LinB and LinA gene in LB medium with inducer and 5Mm γ-HCH respectively, as the substrate was monitored in batch culture. The result was screened by GC analysis(Fig.4). Growth resulted in disappearance of the substrate and simultaneous formation of biomass and γ-PCCH et al. But available columns were limited, hence the intermediate product couldn’t be detected. Through SDS-PAGE analysis, F2A peptide could cleave LinA and LinB(Fig.5). LinB would degrade the intermediate product of γ-HCH and according to theory, the γ-HCH could be degraded to desired low toxic compounds.

Uestclifer2.1.png
Fig.4. E.coli strain MC1061 carried LinA and LinA+F2A+LinB gene incubated in LB medium with γ-HCH(5mM) at 30℃. The concentration of γ-HCH was measured by GC. The bacteria carried gene LinA+F2A+LinB degraded γ-HCH(5mM) but the intermediate product couldn't be detected. Blank control(empty vector) have been added, the concentration of γ-HCH merely remained unchanged.

Uestclifenote11.jpg
FIG.5. SDS-PAGE analysis. Supernatant and sediment collected after breaking cell. Lane1 and lane2 are blank controls, in the lane3 and lane5 LinA and LinB are solved in the buffer; in the lane7 and lane8, F2A peptide cleaved LinA and LinB, both of them are solved. The most of protein, LinA+F2A+LinB, is in the sediment as a kind of inclusion body.

P2A Being An Excellent Linker in Chimeric Protein

As for P2A peptide sequence, it didn’t cleave the DhaA and HheC, and this chimeric protein was linked by P2A peptide. (Fig.6).
DhaA+P2A+HheC gene were expressed in E.coli strain MC1061. Separating supernatant and sediment of the broken cell liquid and used colorimetric method to detect the activity with TCP and 1,3-DCP.(Fig.7). DhaA+P2A+HheC in supernatant had activity of DhaA and HheC enzymes, while in sediment, there wasn't any activity.
HheC has a strict structure. Only if four HheC subunits compose together as a tetramer can it catalyze dehalogenation. Measuring the activity of HheC in chimeric protein DhaA+P2A+HheC is an ideal way to detect the effect of P2A peptide. The purified enzymes, HheC and DhaA+P2A+HheC chimeric protein was isolated by AKTA FPLC. Using substrate 1,3-DCP, HheC specific activity was 6.72(U/mg) and the chimeric protein specific activity is 5.28(U/mg). It indicated that the 2A peptide was an excellent linker to compose the two enzymes together.

Uestclifenote12.jpg
Fig.6 lane7 and lane8 are blank controls, lane1 and lane3 are the expression of DhaA and HheC gene. In the lane5 and lane6, HheC and DhaA bands couldn’t be found,there is DhaA+P2A+HheC chimeric protein band, P2A peptide sequence can’t cleave DhaA and HheC. In the sediment, it is DhaA+P2A+HheC inclusion body.

Uestclifer3.2.png Uestclifer3.3.png
Fig.7 the reaction in buffer(50MmTris-SO3,PH=8.0), after breaking cell, the supernatant and sediment were separated. Blank control was the cell transformed empty vector. Colorimetric method is a way of detecting the concentration of Cl^-(refer to protocol). The solved chimeric protein DhaA+P2A+HheC could degrade 1,3-DCP and TCP. The inclusion protein didn’t have any activity.

Polycistronic Co-expression System Constructed

In polycistronic co-expression system, the quantity of HheC was less than DhaA and LinB is less than LinA(Fig.9). Because the further the distance between coding and promoter is, the less quantity of expression is. That was why we wanted to utilize 2A peptide in prokaryotic system.

Uestclifer4.1.png Uestclifenote22.png
Fig.9 (a) In the lane5 and lane6, LinA and LinB are expressed, according to the bands, the quantity of LinB is less than LinA. (b) In the lane1 and lane2, there are both bands of DhaA and HheC, the quantity of DhaA is more than HheC.

Vectors and Parts

POHC00.jpg pOHC_00 empty modified pBAD vector
POHC01.png pOHC_01 LinA at pBAD vector
POHC02.png pOHC_02 LinB at pBAD vector
POHC03.png pOHC_03 DhaA at pBAD vector
POHC04.png pOHC_04 HheC at pBAD vector
POHC05.png pOHC_05 LinA+F2A+LinB at pBAD vector
POHC06.png pOHC_06 DhaA+P2A+HheC at pBAD vector
POHC07.jpg pOHC_07 LinA+RBS+LinB at pBAD vector
POHC08.jpg pOHC_08 DhaA+RBS+HheC at pBAD vector


Parts we submitted

BBa_K1199041 LinA at pSBC31
BBa_K1199042 LinB at pSBC31
BBa_K1199043 DhaA31 at pSBC31
BBa_K1199044 HheC/W249P at pSBC31
BBa_K1199045 LinA+F2A+LinB at pSBC31
BBa_K1199046 DhaA31+P2A+HheC/W249P at pSBC31
BBa_K1199047 LinA+RBS+LinB at pSBC31
BBa_K1199049 DhaA31+RBS+HheCW249P at pSBC31

Future Work

● Assaying the change of intermediate products


● Improve the activity of DhaA and HheC


● Decreasing inclusion body


● Using P2A to create other chimeric protein

Achievement

https://igem.org/2013_Judging_Form?id=1199

Retrieved from "http://2013.igem.org/Team:UESTC_Life/Results_and_discussion"