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Team:TU Delft-Leiden/WetLab/landmine/cloning
From 2014.igem.org
Landmine Module - Cloning
Final constructs
The final BioBricks desired to be constructed were based on both promoters that are activated in presence of land mine compounds:
yqjF
ybiJ
Combination of yqjF and ybiJ
yqjF promoter
Two final constructs are based on the yqjF promoter alone: BBa_K1316003 and BBa_K1316007 (figures 1 and 2). For both of them the reporter gene mKate2 needs to be present behind the yqjF promoter. As BBa_K1316007 was built on bases of BBa_K1316003, a double transcriptional terminator was designed to be after the mKate2 gene. Otherwise, the RNA polymerase would continue transcribing and some unfinised transcripts of the N-genes could interfere with the normal expression of these genes. Therefore, mKate2 gene was first PCRed out of a comercial plasmid and introduced in front of a Double terminator ( BBa_K823017 ).
The yqjF land mine promoter was PCRed out of the plasmid obtained from Belkin's lab [1] and cloned in front of mKate2.
The construct BBa_K1316007 contains three genes (N-genes) assumed to be involved in the degradation of nitrogene compounds and, therefore, it was hypothesised that they could enhance the action of the used promoters (yqjF and ybiJ). These genes were synthesised "de novo" and cloned behind the double terminator. These genes were designed under the regulation of the Rhamnose inducible promoter, so that the influence of these N-genes can be easily tested.
A more graphical representation of the cloning strategy can be found on figure 3.
ybiJ promoter
Two final constructs are based on the ybiJ promoter alone: BBa_K1316005 and BBa_K1316008. For both of them the reporter gene mKate2 needs to be present behind the yqjF promoter. As BBa_K1316008 was built on bases of BBa_K1316005, a double transcriptional terminator was designed to be after the mKate2 gene. Otherwise, the RNA polymerase would continue transcribing and some unfinised transcripts of the N-genes could interfere with the normal expression of these genes. Therefore, mKate2 gene was first PCRed out of a comercial plasmid and introduced in front of a Double terminator ( BBa_K823017 ).
The yqjF land mine promoter was PCRed out of the plasmid obtained from Belkin's lab [1] and cloned in front of mKate2.
The construct BBa_K1316008 contains three genes (N-genes) assumed to be involved in the degradation of nitrogene compounds and, therefore, it was hypothesised that they could enhance the action of the used promoters (yqjF and ybiJ). These genes were synthesised "de novo" and cloned behind the double terminator. These genes were designed under the regulation of the Rhamnose inducible promoter, so that the influence of these N-genes can be easily tested.
Combination of yqjF and ybiJ
The construct BBa_K1316009 contains two copies of mKate2 regulated with one of the two studied promoters (yqjF and ybiJ) each (figure 7). For this construct, again, having a double transcriptional terminator between both coding sequences is important to prevent the formation of non-desired unfinished transcripts. In this case, the combination p[yqjF]-mKate2-TT-p[ybiJ]-mKate2 (being TT the double terminator) was created by restriction of "p[ybiJ]-mKate2" from BBa_K1316005 using XbaI and PstI and ligating it to BBa_K1316003 cut with SpeI and PstI, as shown in the picture . Nevertheless, having a double terminator behind BBa_K1316005 also allows for the formation of the contruct: p[ybiJ]-mKate2-TT-p[yqjF]-mKate2.
Containing both promoters, this construct aims to trigger a better response in presence of the chemical compounds desired to be detected (2,4-DNT and 1,3-DNB).
References
[1] S. Yagur-Kroll, S. Belkin et al., “Escherichia Coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene”, Appl. Microbiol. Biotechnol. 98, 885-895, 2014.
