Team:TU Delft-Leiden/WetLab/landmine/cloning

From 2014.igem.org

Module Landmine Detection – Cloning

In the wet lab, we made constructs containing the fluorescent protein mKate2 regulated by the ybiJ and yqjF promoters, which are thought to be activated with some chemicals such as 2,4-DNT or 1,3-DNB. Here you can find information with respect to cloning of the BioBricks for the Landmine Detection module.

Module Landmine Detection

Characterization

yqjF promoter

Final constructs

Two final constructs are based on the yqjF promoter: BBa_K1316003 and BBa_K1316007 (figures 1 and 2).

Figure 1: Plasmid containing the BBa_K1316003 BioBrick.

Figure 2: Plasmid containing the BBa_K1316007 BioBrick. "no il rest sites" stands for "no illegal iGEM restriction sites", as the N-genes were designed "de novo" in a way that illegal iGEM restriction sites were avoided.

Cloning Scheme

For both BioBricks BBa_K1316003 and BBa_K1316007 the reporter gene mKate2 needs to be present behind the yqjF promoter. When the RNA polymerase binds to a promoter it keeps on transcribing DNA into mRNA until it falls out due to a transcriptional terminator or due to spontaneous detachment from the DNA. Therefore, a double transcriptional terminator was designed to be after the mKate2 gene. Otherwise, in the construct BBa_K1316007 the RNA polymerase would continue transcribing and some unfinished 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 L-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.

Figure 3: Cloning scheme for the constructs based on the yqjF promoter.

ybiJ promoter

Final constructs

Two final constructs are based on the ybiJ promoter: BBa_K1316005 and BBa_K1316008.

Figure 4: Plasmid containing the BBa_K1316005 BioBrick.

Figure 5: Plasmid containing the BBa_K1316008 BioBrick. "no il rest sites" stands for "no illegal iGEM restriction sites", as the N-genes were designed "de novo" in a way that illegal iGEM restriction sites were avoided.

Cloning Scheme

For both constructs the reporter gene mKate2 needs to be present behind the ybiJ promoter. For the same reason explained for BBa_K1316003 and BBa_K1316007, a double transcriptional terminator was designed to be after the mKate2 gene. Therefore, mKate2 gene was first PCRed out of a comercial plasmid and introduced in front of a Double terminator ( BBa_K823017 ).

The ybiJ 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 the three N-genes that were hypothesised to 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 L-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 6.

Figure 6: Cloning scheme for the constructs based on the ybiJ promoter.

Combination of yqjF and ybiJ

Final construct

The final construct is the BBa_K1316009 BioBrick, and it contains two copies of mKate2 regulated with one of the two studied promoters (yqjF and ybiJ) each (figure 7).

Figure 7: Plasmid containing the BBa_K1316009 BioBrick.

Cloning Scheme

For this construct BBa_K1316009, 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 on figure 8 . 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 at triggering a better response in presence of the chemical compounds desired to be detected (2,4-DNT and 1,3-DNB).

A more graphical representation of the cloning strategy can be found on figure 8.

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.