Difference between revisions of "Team:Nankai/Basic Part"
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<h6> Figure 1. Promoter strength assay in <em>Bacillus amyloliquefaciens</em> NK-1.</h6> | <h6> Figure 1. Promoter strength assay in <em>Bacillus amyloliquefaciens</em> NK-1.</h6> | ||
<p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px; font-weight: bold;">Coding genes</p> | <p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px; font-weight: bold;">Coding genes</p> | ||
| − | <p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px;"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628101">BBa_K1628101</a> (<em>pgsB</em>) and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628102">BBa_K1628102</a> (<em>pgsAC</em>) are genes in <em>pgsBCA</em> operon. <em>pgsB</em> is a gene responsible for γ-PGA synthesis.Protein PgsBCA is a membrane protein and subunit PgsB’s main function is gathering substrate glutamic acid for γ-PGA synthesis (showed in Figure 3). Subunit <em>PgsC</em> is responsible for glutamic acid’s polymerization and subunit <em>PgsA</em> is responsible for the secretion of γ-PGA.We transformed the plasmids pHT01-<em>xylR</em> and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of <em>Bacillus amyloliquefaciens</em> strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of <em>bgaB</em>. As shown in Figure 2, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of <em>bgsB </em>in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in <em>B. amyloliquefaciens</em> NK-1 strain.</p> | + | <p style="position: relative; top: 0px; left: 20px; width: 700px; font-size: 18px; font-family: calibri,Arial, Helvetica, sans-serif; text-align: justify; line-height: 30px;"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628101">BBa_K1628101</a> (<em>pgsB</em>) and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1628102">BBa_K1628102</a> (<em>pgsAC</em>) are genes in <em>pgsBCA</em> operon. <em>pgsB</em> is a gene responsible for γ-PGA synthesis.Protein PgsBCA is a membrane protein and subunit PgsB’s main function is gathering substrate glutamic acid for γ-PGA synthesis (showed in Figure 3). Subunit <em>PgsC</em> is responsible for glutamic acid’s polymerization and subunit <em>PgsA</em> is responsible for the secretion of γ-PGA.We transformed the plasmids pHT01-<em>xylR</em> and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of <em>Bacillus amyloliquefaciens</em> strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of <em>bgaB</em>. As shown in Figure 2, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of <em>bgsB </em>in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in <em>B. amyloliquefaciens</em> NK-1 strain.Funtion of part <em>pgsB</em> and <em>pgsCA</em> were validated through fermentation experiment (showed in Figure 2). Wild type <em>Bacillus amyloliquefaciens </em>NK-1 strain could produce 3-4g/L γ-PGA after 48 hours of fermentation. In previous work in our laboratory, we also tested heterologous expression of γ-PGA in <em>E. coli</em>. We constructed different expression vectors containing <em>pgsBCA</em> genes and transformed them separately into E.coli JM109. The average production of γ-PGA in <em>E. coli</em> JM109 is around 0.5g/L</p> |
<img src="https://static.igem.org/mediawiki/2015/4/48/Parts_figure_pgsABC.jpeg"> | <img src="https://static.igem.org/mediawiki/2015/4/48/Parts_figure_pgsABC.jpeg"> | ||
| − | <h6> Figure 2. | + | <h6> Figure 2. γ-PGA produced by<em> B. amyloliquefaciens</em> NK-1</h6> |
<img src="https://static.igem.org/mediawiki/2015/2/25/Parts_figure3.jpeg"> | <img src="https://static.igem.org/mediawiki/2015/2/25/Parts_figure3.jpeg"> | ||
<h6>Figure 3. The synthetic pathway of γ-PGA</h6> | <h6>Figure 3. The synthetic pathway of γ-PGA</h6> | ||
Revision as of 14:30, 18 September 2015
Basic Parts
Promoters
According to our promoter strength assay (showed in Figure 1), BBa_K1628001 (Pbca), BBa_K1628003 (BJ27UP), BBa_K1628004 (C2up), BBa_K1628005 (A2up), BBa_K1628006 (P43), and BBa_K1628007 (PamyA) show different properties. Promoter Pbca is the original promoter of pgsBCA operon.Promoter BJ27UP C2up and A2up are an artificially synthesized promoters. Promoter P43 is a strong promoter in Bacillus subtilis 168. Promoter PamyA is a strong promoter in Bacillus amyloliquefaciens LL3.
Judging from the promoter strength assay, BBa_K1628004 (C2up) is the strongest promoters we used in our project and BBa_K1628007 (PamyA) is the second strongest promoter in our project. The strength of BBa_K1628001 (Pbca) is very weak. While the strength of BBa_K1628003 (BJ27UP) is stronger than Pbca, it is still too weak compared with other promoters. What's more, BBa_K1628005 (A2up) is a very strong promoter and BBa_K1628006 (P43) is a weak promoter.
Figure 1. Promoter strength assay in Bacillus amyloliquefaciens NK-1.
Coding genes
BBa_K1628101 (pgsB) and BBa_K1628102 (pgsAC) are genes in pgsBCA operon. pgsB is a gene responsible for γ-PGA synthesis.Protein PgsBCA is a membrane protein and subunit PgsB’s main function is gathering substrate glutamic acid for γ-PGA synthesis (showed in Figure 3). Subunit PgsC is responsible for glutamic acid’s polymerization and subunit PgsA is responsible for the secretion of γ-PGA.We transformed the plasmids pHT01-xylR and pCB-Pxyl into the NK-1 strain, to verify the activity of metabolic toggle switch (see it on our wiki). Fresh colonies of Bacillus amyloliquefaciens strains (NK-1 strain containing plasmids pHT01-xylR and pCB-Pxyl and the control NK-1 strain containing plasmids pHT01 and pCB-Pxyl) were first cultured overnight in test tubes containing 5 mL LB liquid and then inoculated into 100 mL fresh fermentation medium. We added 1mM IPTG into the medium after 12h of cultivation. The β-galactosidase activity were measured at 12h, 18h, 24h, 30h, 36h, 42h to test the effect of metabolic toggle switch on the expression of bgaB. As shown in Figure 2, β-galactosidase enzyme activity dropped considerably after 30 hours of fermentation. The inhibited expression of bgsB in experiment group (NPP+IPTG) indicated that the metabolic toggle switch we constructed is functional in B. amyloliquefaciens NK-1 strain.Funtion of part pgsB and pgsCA were validated through fermentation experiment (showed in Figure 2). Wild type Bacillus amyloliquefaciens NK-1 strain could produce 3-4g/L γ-PGA after 48 hours of fermentation. In previous work in our laboratory, we also tested heterologous expression of γ-PGA in E. coli. We constructed different expression vectors containing pgsBCA genes and transformed them separately into E.coli JM109. The average production of γ-PGA in E. coli JM109 is around 0.5g/L
Figure 2. γ-PGA produced by B. amyloliquefaciens NK-1
Figure 3. The synthetic pathway of γ-PGA
