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Commit 74e8e54d authored by Yangyang Kong's avatar Yangyang Kong
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<img class="sub-header-logo" src="https://static.igem.wiki/teams/4515/wiki/banner.jpg" />
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<div class="sub-page-bg">
<div class="sub-page-content">
<div class="content-title">Contribution</div>
<div class="title blue-title">Overview</div>
<div class="article-content ">
Our composite part BBa_K4515012 is the N-butanol pathway we used in Streptococcus Brevis ATCC367. It is improved
based on the existing part BBa_K1462040, this is a biological part submitted by iGEM14_SCUT in 2014, with only DNA
sequence information and simple text description information. Because the tolerance of Clostridium bacteria to
N-butanol is not good enough for large-scale production. Based on this problem, we chose Streptococcus Brevis
ATCC367, a lactobacillus with better N-butanol tolerance that has been isolated by researchers, as our host strain
in this project. Our team carried out a comprehensive characterization of this part in the laboratory, adding data
from fermentation testing to dedicate its function of producing N-butanol. This information can be a good
reference for future iGEM teams working on improving the yield of N-butanol.<br />
In addition, through literature research, we developed an N-butanol biosynthesis pathway,
Pcrt-crt-ter-hbd-Pthl-thl-opt and constructed these genes in the plasmid. What's more, we transferred the
recombinant plasmid into Streptococcus Brevis ATCC367 to establish an N-butanol-producing platform and measured
the yield of N-butanol. Then, by detecting the growth curve of Streptococcus Brevis ATCC367 transformants, it was
further confirmed that Streptococcus Brevis ATCC367 has better tolerance for N-butanol and could be used to
produce N-butanol in factories in the future. We upload the DNA sequence information and basic introduction
information in the registry of standard biological parts to provide more choices of N-butanol-producing for future
iGEM teams.
</div>
<div class="title blue-title">Add new experimental data to an existing Part BBa_K1462040, crt</div>
<div class="article-content ">Gene crt encodes 3-Hydroxybutyryl-CoA dehydratase, which converts 3-Hydroxybutyryl-CoA
to Crotonyl-CoA, the third step of the N-butanol pathway. </div>
<div class="sub-title">a) Construction of N-butanol biosynthesis pathway with gene crt</div>
<div class="article-content ">Gene ctr was promoted by the Pcrt promoter and other related genes, thlA, hbd, and
ter, were all amplified from the Lactobacillus Brevis ATCC824 genomic DNA through PCR. The DNA sequences of the
Pcrt-crt-ter-hbd-Pthl-thl-opt was inserted into the ApaI and BglII sites of the pIB184 vector, respectively. The
certificate of recombinant plasmid sequencing results is as Figure 1.</div>
<div class="img-wrap no-margin">
<img class="w-80" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-contribution01.jpg" />
<span>Figure 1. The results of the sequencing data mapped to the plasmids</span>
</div>
<div class="sub-title">b) Functional Test</div>
<div class="article-content">
To confirm if the Pcrt-crt-ter-hbd-Pthl-thl-opt system worked well in the host strain Streptococcus Brevis
ATCC367, we also measured the yield of N-butanol through gas chromatography. As shown in Figure 2, the yield of
N-butanol is increasing with an increased time of fermenting.
</div>
<div class="img-wrap no-margin">
<img class="w-80" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-contribution02.jpg" />
<span>Figure 2. After pLY15-opt was transformed into Streptococcus Brevis, N-butanol production of ply15-opt
strain was measured at different times (48h, 69h, 95h, and 159h)</span>
</div>
<div class="title blue-title">Add new information to the Part BBa_K4515012, BBa_K4515010, and BBa_K4515014</div>
<div class="sub-title">a) BBa_K4515012, Pcrt-crt-ter-hbd-Pthl-thl-opt:</div>
<div class="article-content">
Genes thlA, crt, hbd, and ter, play important roles in the N-butanol biosynthesis pathway. Those genes were
codon-optimized. Gene thlA is coding for acetyl-CoA acetyltransferase and converts Acetyl-CoA into Acetoacetyl-CoA
in the N-butanol biosynthesis pathway. Gene hbd, encodes β-Hydroxybutyryl-CoA dehydrogenase and converts
Acetoacetyl-CoA into 3-Hydroxybutyryl-CoA. Gene crt encodes 3-Hydroxybutyryl-CoA dehydratase, which converts
3-Hydroxybutyryl-CoA to Crotonyl-CoA, the third step of the N-butanol pathway. In this part, genes crt, ter and
hbd were promoted by Pcrt promoter, gene thl was promoted by Pthl promoter, and these DNA fragments were ligated
in order into pIB184 vector.
</div>
<div class="sub-title">b) BBa_K4515010, pIB184-vector</div>
<div class="article-content">pIB184-vector is an E. coli - Streptococci shuttle plasmid for gene expression in
streptococci with P23 promoter. This plasmid is a low-copy plasmid and Erythromycin resistance can be used to
screen the correct colony in bacteria. This vector contains MCS-A. The backbone of this vector is based on pOri23.
</div>
<div class="sub-title">c) BBa_K4515014, pLY15-opt</div>
<div class="article-content">
This composite part is the recombinant plasmid constructed by Pcrt-crt-ter-hbd-Pthl-thl-opt fusion DNA fragment
(BBa_K4515012) and pIB184-vector (BBa_K4515010). This plasmid could be transferred into Streptococcus Brevis
ATCC367 to produce N-butanol.<br />
Above all, we look forward to the future iGEM team making new additions, explorations, and explanations to our
biological components.
</div>
<div class="title blue-title">Reference</div>
<div class="article-content">
1. 张云贤, 张华西, 余维新, 李杰灵, & 谭平华. (2015). 正丁醇的合成进展简述. 2015 中国化工学会学术年会. <br />
2. Li, J., Zhao, J. B., Zhao, M., Yang, Y. L., Jiang, W. H., & Yang, S. (2010). Screening and characterization of
butanol-tolerant micro-organisms. Letters in applied microbiology, 50(4), 373–379.
https://doi.org/10.1111/j.1472-765X.2010.02808.x <br />
3. Berezina, O. V., Zakharova, N. V., Brandt, A., Yarotsky, S. V., Schwarz, W. H., & Zverlov, V. V. (2010).
Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Applied microbiology and
biotechnology, 87(2), 635–646. https://doi.org/10.1007/s00253-010-2480-z <br />
4. Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression
of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biot 77:1305–1316.
https://doi.org/10.1007/s00253-007-1257-5 <br />
5. Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by Clostridia. In: Poole RK (ed) Advances
in Microbial Physiology, vol 39. pp 31–130 <br />
6. Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium-acetobutylicum. Appl Environ Microb
50:1165–1170<br />
7. Biswas I, Jha JK, Fromm N. (2008) Shuttle expression plasmids for genetic studies in Streptococcus mutans.
Microbiology (Reading). Aug;154(Pt 8):2275-2282. doi: 10.1099/mic.0.2008/019265-0.
</div>
</div>
</div>
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{% block title %}Contribution{% endblock %}
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<img class="sub-header-logo" src="https://static.igem.wiki/teams/4515/wiki/banner.jpg" />
{% endblock %}
{% block page_content %}
<div class="sub-page-bg">
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