Difference between revisions of "Team:SKLMT-China/Design"
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<p>From the very beginning of our project is to find standard promoter sequence. All the promoter sequence(70bp) is taken from the upstream region of a coding sequence (exactly upstream of its ATG base).And then, construct plasmid containing a promoter sequence and the reporter gene. </p> | <p>From the very beginning of our project is to find standard promoter sequence. All the promoter sequence(70bp) is taken from the upstream region of a coding sequence (exactly upstream of its ATG base).And then, construct plasmid containing a promoter sequence and the reporter gene. </p> | ||
<span class="image fit"> | <span class="image fit"> | ||
| − | <img class="lb" src="https://static.igem.org/mediawiki/2018/a/ab/T--SKLMT-China--designfig1.png" alt="Fig.1 standard promoter sequence in the project" /> | + | <img class="lb" src="https://static.igem.org/mediawiki/2018/a/ab/T--SKLMT-China--designfig1.png" data-src="https://static.igem.org/mediawiki/2018/a/ab/T--SKLMT-China--designfig1.png" alt="Fig.1 standard promoter sequence in the project" /> |
</span> | </span> | ||
<p>In order to obtain an artificial promoter library suitable for cloning, promoter sequence was added a chloramphenicol selection marker. The linier DNA fragment is prepared through serious PCR, by which the chloramphenicol combined with 70bp standard promoter sequence is flanking with homology arms of the vector. We constructed the plasmids by LCHR.</p> | <p>In order to obtain an artificial promoter library suitable for cloning, promoter sequence was added a chloramphenicol selection marker. The linier DNA fragment is prepared through serious PCR, by which the chloramphenicol combined with 70bp standard promoter sequence is flanking with homology arms of the vector. We constructed the plasmids by LCHR.</p> | ||
<span class="image fit"> | <span class="image fit"> | ||
| − | <img class="lb" src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017125133%21T--SKLMT-China--TangFei.svg" alt="Fig.2 promoter library construction design" /> | + | <img class="lb" src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017125133%21T--SKLMT-China--TangFei.svg" data-src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017125133%21T--SKLMT-China--TangFei.svg" alt="Fig.2 promoter library construction design" /> |
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<span class="image fit"> | <span class="image fit"> | ||
| − | <img class="lb" src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017131652%21T--SKLMT-China--TangFei.svg" data-src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017131652%21T--SKLMT-China--TangFei.svg" alt="Fig.3 Firefly luciferase assay kit" /> | + | <img class="lb" src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017131652%21T--SKLMT-China--TangFei.svg" data-src="https://static.igem.org/mediawiki/2018/archive/2/21/20181017131652%21T--SKLMT-China--TangFei.svg" alt="Fig.3 Firefly luciferase assay kit" |
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<p> Since predecessors have less research on the promoter collection and characterization of <latin>P. fluorescence </latin>we have done an innovative work and set a standard for the promoter strength in<latin> P.fluorescence</latin>. based on the data, we established a modelling work to finger out the link between promoter sequence and strength.</p> | <p> Since predecessors have less research on the promoter collection and characterization of <latin>P. fluorescence </latin>we have done an innovative work and set a standard for the promoter strength in<latin> P.fluorescence</latin>. based on the data, we established a modelling work to finger out the link between promoter sequence and strength.</p> | ||
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<p>In addition to the formation of the promoter library, we also constructed a nicotine-degrading engineered bacteria using pf-5 as the chassis.</p> | <p>In addition to the formation of the promoter library, we also constructed a nicotine-degrading engineered bacteria using pf-5 as the chassis.</p> | ||
<span class="image fit"> | <span class="image fit"> | ||
| − | <img class="lb" src="https://static.igem.org/mediawiki/2018/2/21/T--SKLMT-China--TangFei.svg" alt="Fig.4 nicotine degradation experiment design" data-src="https://static.igem.org/mediawiki/2018/2/21/T--SKLMT-China--TangFei.svg" alt="Fig.4 nicotine degradation experiment design"width | + | <img class="lb" src="https://static.igem.org/mediawiki/2018/2/21/T--SKLMT-China--TangFei.svg" alt="Fig.4 nicotine degradation experiment design" data-src="https://static.igem.org/mediawiki/2018/2/21/T--SKLMT-China--TangFei.svg" alt="Fig.4 nicotine degradation experiment design" style="width: 80%"/> |
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Revision as of 13:28, 17 October 2018
introduction
LCHR
LLHR
Design
outline
Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. This year the team SKLMT-China wants to deal with the environmental problem using a new extraordinary chassis bacteria,
Promoter llibrary construction
From the very beginning of our project is to find standard promoter sequence. All the promoter sequence(70bp) is taken from the upstream region of a coding sequence (exactly upstream of its ATG base).And then, construct plasmid containing a promoter sequence and the reporter gene.
In order to obtain an artificial promoter library suitable for cloning, promoter sequence was added a chloramphenicol selection marker. The linier DNA fragment is prepared through serious PCR, by which the chloramphenicol combined with 70bp standard promoter sequence is flanking with homology arms of the vector. We constructed the plasmids by LCHR.
The strength of different promoter was characterized by a reporter gene,
Since predecessors have less research on the promoter collection and characterization of
Nicotine degradation
In addition to the formation of the promoter library, we also constructed a nicotine-degrading engineered bacteria using pf-5 as the chassis.
We completed the whole molecular clone process by Red/ET reconbination technology. First of all, digeste the genome of
Second, in order to get this plasmid replicated in
Next, we’ve found there is a part of chlorampheol
Next, we’ve found there is a part of chlorampheol
Finally, we got our promoter4,5and11 segments through PCR using PAGE-purified oligonucleotides containing 20-nt homology arms flanking the target pSB1C3-promoter-cm plasmids and 20-nt standard PCR primers at 3’end. At last, we transferred these three promoter segments into