Difference between revisions of "Part:BBa K1796015"
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<h2>iGEM2018_Nanjing China Experiment</h2> | <h2>iGEM2018_Nanjing China Experiment</h2> | ||
− | + | <p>This year our team used the nitrogen fixation gene cluster from Paenibacillus polymyxa CR1, which shares a close biological relationship with Paenibacillus sp.WLY78, so the data can provide some reference to this part.</p> | |
− | <p> | + | <p>To test whether the nitrogen fixation gene cluster could express in gram-negative <em>E. coli</em> JM109 , pUC57-<em>nif </em>was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in <em>nif</em> cluster (P<em>nif</em>) were firstly tested in JM109 by fusing Dronpa as the reporter. T5 promoter (BBa_M50075) severed as control. As shown in Figure 1b, compared with T5 promoter, P<em>nif </em>was much stronger in driving the expression of RFP and its expression pattern was constitutive. Transcriptional analysis was carried out afterward. As shown in Figure 2, P<em>nif</em> was strong enough to drive the expression of each structure gene in the <em>nif</em> cluster including <em>nif</em>B though with different relative expression level.</p> |
− | + | <p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a)Engineered E. coli cells with nitrogenase<br /> | |
− | <p> | + | 1b)Fluorescence intensity detemination]] </p> |
− | + | <p> [[File:T--Nanjing-China--qRT-PCR.jpg|800px|thumb|center|Figure 2. Expression profiles of each structure gene in the nif cluster that overexpressed in engineered E.coli JM109 (EJNC). E.coli JM109 (EJ) severs as control and relative expression compared to the housekeeping gene 16S rRNA is shown. N.D. represent not ditected.]]</p> | |
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Latest revision as of 10:45, 16 October 2018
complete line of nif cluster
whole line of nif cluster,contain Pnif,nifBHDKENXV,hesA
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2163
Illegal EcoRI site found at 6951
Illegal PstI site found at 2689
Illegal PstI site found at 6597
Illegal PstI site found at 7295
Illegal PstI site found at 7866
Illegal PstI site found at 7937
Illegal PstI site found at 8743
Illegal PstI site found at 8983 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2163
Illegal EcoRI site found at 6951
Illegal PstI site found at 2689
Illegal PstI site found at 6597
Illegal PstI site found at 7295
Illegal PstI site found at 7866
Illegal PstI site found at 7937
Illegal PstI site found at 8743
Illegal PstI site found at 8983 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2163
Illegal EcoRI site found at 6951
Illegal BglII site found at 3655
Illegal BglII site found at 10747
Illegal BamHI site found at 10570 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2163
Illegal EcoRI site found at 6951
Illegal PstI site found at 2689
Illegal PstI site found at 6597
Illegal PstI site found at 7295
Illegal PstI site found at 7866
Illegal PstI site found at 7937
Illegal PstI site found at 8743
Illegal PstI site found at 8983 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2163
Illegal EcoRI site found at 6951
Illegal PstI site found at 2689
Illegal PstI site found at 6597
Illegal PstI site found at 7295
Illegal PstI site found at 7866
Illegal PstI site found at 7937
Illegal PstI site found at 8743
Illegal PstI site found at 8983
Illegal NgoMIV site found at 5942
Illegal NgoMIV site found at 6189
Illegal NgoMIV site found at 7624
Illegal AgeI site found at 1136
Illegal AgeI site found at 2096
Illegal AgeI site found at 2451
Illegal AgeI site found at 3680
Illegal AgeI site found at 4362
Illegal AgeI site found at 4735
Illegal AgeI site found at 5202 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2268
iGEM2018_Nanjing China Experiment
This year our team used the nitrogen fixation gene cluster from Paenibacillus polymyxa CR1, which shares a close biological relationship with Paenibacillus sp.WLY78, so the data can provide some reference to this part.
To test whether the nitrogen fixation gene cluster could express in gram-negative E. coli JM109 , pUC57-nif was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in nif cluster (Pnif) were firstly tested in JM109 by fusing Dronpa as the reporter. T5 promoter (BBa_M50075) severed as control. As shown in Figure 1b, compared with T5 promoter, Pnif was much stronger in driving the expression of RFP and its expression pattern was constitutive. Transcriptional analysis was carried out afterward. As shown in Figure 2, Pnif was strong enough to drive the expression of each structure gene in the nif cluster including nifB though with different relative expression level.