Team:Edinburgh/Project/Results/Metal promoters Results

From 2013.igem.org

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Using the protocol found in the [https://2013.igem.org/Team:Edinburgh/GenBrick/GenablerAppendix appendix], a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004). More information on the structure of the assembly can be found in the [https://2013.igem.org/Team:Edinburgh/GenBrick/GenbrickAction GenBrick in action section].
Using the protocol found in the [https://2013.igem.org/Team:Edinburgh/GenBrick/GenablerAppendix appendix], a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004). More information on the structure of the assembly can be found in the [https://2013.igem.org/Team:Edinburgh/GenBrick/GenbrickAction GenBrick in action section].
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The assembly was used as part of the WastED project of EdiGEM 2013. Two transformations were made and plated on IPTG + chloramphenicol plates and left overnight. In one plate, 4 out of 9 colonies were fluorescent; the other five had religated and were red. The other plate had a success rate of 8 out of 20 (the competent cells were concentrated x10 before plating). Figure 2 shows the assembly fluorescing under UV when no iron was present and Figure 3 shows the gel of restriction-digestion gel using PstI and XbaI.
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''' ''Figure 2.'' ''' Fur box Assembly. After transforming competent cells with the assembly and leaving overnight on an IPTG + chloramphenicol plate, 2 green fluorescent colonies were restreaked. A gel was performed to show the parts had correctly assembled.
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''' ''Figure 3'' ''' Fur box digest. On the left is the ladder, the first column is the undigested assembly which shows only one band (the second lower band is an artifact due to leakage). The second column contains the digested assembly. The size of PLac_LacZ + linkers + GFP was correct.
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Revision as of 19:45, 4 October 2013

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Fur Box Assembly


Using the protocol found in the appendix, a 3-part construct was assembled with an acceptor vector pSB1C3 (BBa_K1122009), a Lac promoter and truncated gene called PLac_LacZ (BBa_J33207) optimised for GenBrick and a fluorescent protein, GFP (BBa_K1122004). More information on the structure of the assembly can be found in the GenBrick in action section.


The assembly was used as part of the WastED project of EdiGEM 2013. Two transformations were made and plated on IPTG + chloramphenicol plates and left overnight. In one plate, 4 out of 9 colonies were fluorescent; the other five had religated and were red. The other plate had a success rate of 8 out of 20 (the competent cells were concentrated x10 before plating). Figure 2 shows the assembly fluorescing under UV when no iron was present and Figure 3 shows the gel of restriction-digestion gel using PstI and XbaI.

Furbox1.png

Furbox2.png

Figure 2. Fur box Assembly. After transforming competent cells with the assembly and leaving overnight on an IPTG + chloramphenicol plate, 2 green fluorescent colonies were restreaked. A gel was performed to show the parts had correctly assembled.

Figure 3 Fur box digest. On the left is the ladder, the first column is the undigested assembly which shows only one band (the second lower band is an artifact due to leakage). The second column contains the digested assembly. The size of PLac_LacZ + linkers + GFP was correct.



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This iGEM team has been funded by the MSD Scottish Life Sciences Fund. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of MSD
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