Difference between revisions of "Team:Bielefeld-CeBiTec/Results/unnatural base pair/uptake and biosynthesis"

Revision as of 14:52, 28 October 2017

Uptake and Biosynthesis

Computational Analysis of PtNTT2

Zhang et al. used an N-terminal truncated version of PtNTT2, lacking the first 65 amino acids, since they observed some kind of toxicity resulting from the native N-terminal sequence (Zhang et al., 2017). For our project, we analyzed the amino acid sequence of PtNTT2 using the prediction software Phobius (Käll et al., 2007). Using Phobius, we analyzed the signal peptide and the transmembrane topology of PtNTT2. The analysis revealed that the native signal peptide is formed by amino acids 1 30, which means that Zhang et al. removed more than the native signal peptide for their experiment. The results of the prediction are shown in Figure X. Analysis of the transmembrane topology of the transporter, which is integrated into the plastid membrane in its native algal cell, shows iterative non-cytoplasmatic, transmembrane and cytoplasmic regions. The topology might indicate, that the transporter will be integrated into the inner membrane when expressed in E. coli.

Figure (1): Results of the analysis of PtNTT2 using Phobius.
The 30 first amino acids are clearly recognized as a signal peptide. Ten transmembrane domains are predicted.

Plasmid Design

For the analysis and characterization of PtNTT2, a total of eleven plasmids were designed and cloned based on initial research and the computational analysis. The coding sequence of PtNTT2 was codon optimized using the IDT Codon Optimization Tool [REF] and ordered as two gBlocks. Using overlap extension PCR, the two gBlocks were put together and inserted into pSB1C3 using Gibson Assembly. The truncated versions of the transporter as well as the versions with new signal peptides were constructed using primers and Gibson assembly. Based on BBa_B0034 and BBa_K2082221 the fusion proteins were designed and cloned. A schematic overview of the design of the different plasmids is shown in Figure 2. All plasmids except pSB1C3-PtNTT2 were tagged with the same cMyc-GFP construct for imaging and western blots.

Figure (2): Schematic overview of the design of the different transporter variants.
The lacUV5 promotor was used together with a strong RBS (BBa_B0034) for all parts. All variants except for pSB1C3-PtNTT2 were also tagged with GFP (BBa_E0040). cMyc was used as a linker (based on BBa_K2082221).

Cultivations of the Different PtNTT2 Variants

Given that Zhang et al., 2017 reported some kind of toxicity resulting from the N-terminal sequence of PtNTT2, we first investigated if we could also observe the same toxicity associated with the N-terminal sequence. We also tested whether our own versions of the transporter result in better growth and reduced toxicity compared to the native transporter. Therefore, after cloning the plasmids in E. coli DH5α and verifying the correct assembly via sequencing, all plasmids were transformed into E. coli BL21(DE3). The presence of the correct plasmids was again verified by colony PCR. The first cultivations were carried out in shake flasks in LB media. A total cultivation volume of 50 mL was used. The cultures were incubated at 37 °C and 180 rpm. All cultures were inoculated with an OD₆₀₀ of 0.01. OD₆₀₀ was measured every hour during lag and stationary phase and every 30 minutes during the exponential phase. The optical density was measured using an Eppendorf Photometer and standard cuvettes. E. coli BL21(DE3) without a plasmid and E. coli BL21(DE3) harboring pSB1C3-PtNTT2 were used as negative controls. Two biological replicates of each strain were tested and three technical replicates were measured for each timepoint.

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-			<img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c6/T--Bielefeld-CeBiTec--Phobius_prediction.jpeg">
+			<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f7/T--Bielefeld-CeBiTec--Phobius-Prediction.jpeg">
 			<p class="figure subtitle"><b>Figure (1): Results of the analysis of <i>Pt</i>NTT2 using Phobius.</b><br> The 30 first amino acids are clearly recognized as a signal peptide. Ten transmembrane domains are predicted.</p>
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+	<div class="content">
+		<!-- Ueberschriften -->
+		<h3> Cultivations of the Different <i>Pt</i>NTT2 Variants </h3>
+		<article>Given that Zhang <i>et al.</i>, 2017 reported some kind of toxicity resulting from the N-terminal sequence of <i>Pt</i>NTT2, we first investigated if we could also observe the same toxicity associated with the N-terminal sequence. We also tested whether our own versions of the transporter result in better growth and reduced toxicity compared to the native transporter. Therefore, after cloning the plasmids in <i>E.&nbsp;coli</i> DH5α and verifying the correct assembly via sequencing, all plasmids were transformed into <i>E.&nbsp;coli</i> BL21(DE3). The presence of the correct plasmids was again verified by colony PCR.
+		The first cultivations were carried out in shake flasks in LB media. A total cultivation volume of 50 mL was used. The cultures were incubated at 37 °C and 180 rpm. All cultures were inoculated with an OD<sub>600</sub> of 0.01. OD<sub>600</sub> was measured every hour during lag and stationary phase and every 30 minutes during the exponential phase. The optical density was measured using an Eppendorf Photometer and standard cuvettes. <i>E.&nbsp;coli</i> BL21(DE3) without a plasmid and <i>E.&nbsp;coli</i> BL21(DE3) harboring pSB1C3-PtNTT2 were used as negative controls. Two biological replicates of each strain were tested and three technical replicates were measured for each timepoint.
+</article>
+	</div>
 </div>