Difference between revisions of "Part:BBa K1465202"

Latest revision as of 12:28, 19 October 2014

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Halothiobacillus neapolitanus

The Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Halothiobacillus neapolitanus can bind CO₂ by catalyzing the reaction of ribulose 1,5-bisphosphate to 3-phosphoglycerate. The characterization of this part can be found here BBa_K1465213.

Usage and Biology

The Ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) is the most abundant enzyme in the world. Because of its key role in carbon fixation metabolism, it is found in nearly all autotrophic organisms like plants, but also in cyanobacteria and photosynthetic bacteria in high concentrations (Andersson, 2008). RuBisCO catalyses the fixation of atmospheric carbon dioxide by generating two tricarbohydrates out of one pentacarbohydrate. This reaction is part of the Calvin Cycle. It can be stated that the RuBisCO is responsible for conversion of carbon dioxide in biomass or with other words for incorporation of inorganic carbon dioxide to form organic molecules. To give some numbers, more than 10¹¹ tons of atmospheric carbon dioxide are fixated per year baesd on RuBisCO activity (Field et al., 1998).

Figure 1: Catalyzed reaction by the RuBisCO. Ribulose-1,5-bisphosphate and carbon dioxide are converted to two molecules of 3-phosphoglycerate.

RuBisCO catalyses the rate limiting step in the Calvin cycle. RuBisCO catalyses the fixation of one molecule carbon dioxide to ribulose-1,5-bisphosphate (RuBP), a pentacarbohydrate. The product is unstable and decays directly into two molecules of 3-D-phosphoglycerate (3-PGA)(Andersson, 2008; Parikh et al. 2006). The reaction is shown in Figure 1. 3-PGA is further converted in the Calvin cycle to glycerinaldehyde-3-phosphate. This is an essential intermediate in the central metabolism, as it plays a central role in glycolysis and gluconeogenesis.

Beside the carbon fixation reaction of RuBisCO, the enzyme catalyses numerous side reactions. An alternative substrate to carbon dioxide is atmospheric oxygen. When the oxygenation of RuBP is catalyzed instead of the carboxylation, the product is one molecule of 3-PGA and one molecule of 2-phosphoglycolate. 2-phosphoglycolate has only limited use for the metabolism of the cells and the fixed carbon has to be regenerated by a metabolic pathway called photorespiration, a high energy consuming pathway. In photorespiration, two molecules of 2-phosphoglycolate are split up into one molecule of 3-PGA and one molecule of carbon dioxide. 3-PGA can enter the Calvin cycle, whereas CO₂ is a molecule with a low energy content. Because of the oxygenation side reaction the efficiency of the carbon dioxide fixation rate of RuBisCO is reduced about 20 - 50 % (Andersson, 2008; Mann, 1999).
The carboxylation/ oxygenation of RuBP catalyzed by RuBisCO is a multiple step reaction. In detail, the first step is activation of the RuBisCO by carbamylation of the amino group of a lysine in the active centre. The activated RuBisCO is then stabilized by magnesium ions, a cofactor for enzyme activity. In the carboxylation/ oxygenation of Ribulose-1,5-bisphosphate the first step is enolisation of the substrate and enol-RuBP is build up. The enediolate reacts then in an irreversible reaction with either carbon dioxide or oxygen. This reaction determines the specificity and the rate of carbon dioxide fixation as well as the efficiency. If carbon dioxide is bound to the enediolate, the unstable intermediate is protonated and hydrated to build up two molecules of 3-PGA. If oxygen is bound to the enediolate, the intermediate decomposes directly in 3-phosphoglycerate and 2-phosphoglycolate. (Andersson, 2008; Spreitzer, Salvucci 2002)
The competing reaction between CO₂ and O₂ and the resulting oxygenation side reaction limits the efficiency of RuBisCO. The efficiency is often quantified by a specificity factor. This is the ratio of the catalytic efficiency of carboxylation to oxygenation, described by the maximal velocities of carboxylation and oxygenation, and the Michaelis-Menten constants for carbon dioxide and oxygen. (Andersson, 2008; Jordan, Ogren 1981; Spreitzer, Salvucci 2002) The specificity factors of various RuBisCO enzymes differ significantly depending on the host organism of the RuBisCO. Bacteria have low specificity factors in comparison to higher plants or algae. As there exist an inverse correlation between turnover rate (for carboxylation) and specificity factor, Bacteria have low specificity factors, but high turnover rates. Higher organism are characterized by high specificity factors and low turnover rates. (Andersson, 2008; Jordan, Ogren 1981)
RuBisCO is a multiprotein enzyme, which consists of two types of subunits, the large (L) subunit (50-55 kDa) and the small (S) subunit (12-18 kDa). The most common form of RuBisCO (form I or form IA) consists of eight large subunits, which form dimers, and eight small subunits. Together they form a hexadimeric structure. Form I occurs in most autotrophic bacteria, algae and higher plants. The large subunit is the catalytic one, and the small subunit is not essential for catalysis. The octamer of the large subunit still remains its carboxylation activity. RuBisCO form II or form IB is found in some chemoautotrophic bacteria and in dinoflagellates. This form is characterized by the abscence of the small subunits. (Andersson, 2008; Spreitzer, Salvucci 2002)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1366
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 809
Illegal BamHI site found at 1490
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Overview

For the verification of RuBisCo expression, we analyzed protein expression of E. coli KRX containing the construct P_T7cbbLS_{H. neap.} and as a second verification, protein expression using the construct BBa_K1465213. Cultivations were carried out as described in Cultivation for Expression of recombinant proteins.

SDS-Pages

Samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE. The results are shown in Figure 2 and 3.

Figure 2: Protein expression of RuBisCO_{H. neap.} over time by E. coli KRX carrying P_T7cbbLS_{H. neap.} after induction with 0.1 % rhamnose.

Figure 3: Protein expression over time of RuBisCO_{H. neap.} by E. coli carrying BBa_K1465213 after induction with 0.5 mM IPTG.

In both SDS-PAGEs there is a clearly increasing band over the duration of the cultivation. Analysis with MALDI-TOF proved that the band corresponding to a size of a little less than 55 kDa is the large subunit of RuBisCO from Halothiobacillus neapolitanus. The analysis was done via tryptic digestion and an in silico comparison of the measured peptide masses to the predicted peptide masses. For RuBisCO expressed under control of the T7 promoter seven matching peptide masses were found, the sequence coverage was 15.2 % (MS) and 15.2 % (MS/MS). For RuBisCO expressed via the P_tac promoter six matching peptide masses were found and the sequence coverage was 13.5 % (MS) and 13.5 % (MS/MS). The small unit of the RuBisCO could only be identified via MALDI-TOF in the samples expressed under the control of the T7 promoter. The analysis was performed as described above, showing three matching peptide masses and a sequence coverage of 35.5 % (MS) and 12.7 (MS/MS). The reason for the problem in identifying the small subunit stems from its small size of 12.8 kDa, making it hard to find in the SDS-PAGE. Still, these results correspond to the verification of protein expression from the plasmid pHnCBS1D which we used as the basis for purifying of carboxysomes. Expression of this plasmid gave only in one of three experiments the proof of expression the small subunit.

In vitro assay

For the verification of RuBisCO activity, we performed an in vitro assay measuring variances for ribulose-1,5-bisphosphate and 3-phosphoglycerate, substrate and product of the RuBisCo, in the cell extract of KRX wildtype and KRX carrying the construct P_T7 cbbLS_H.neap.. The method for the in vitro assay is described in RuBisCO activity assay and the measuring via HPLC in the protocol for HPLC.
The first measurement with HPLC was made to identify substrate and product of the RuBisCO, Ru-BP and 3-PGA. Therefore, standards containing just one of the substances were measured (Figure 4). The substances are clearly separable with a retention time of 14.4 min for Ru-BP and 12,6 min for 3-PGA.

Figure 4: Standards for the RuBisCO activity assay. The first measurement was ribulose-1,5-bisphosphate, the second was 3-phosphoglycerate.

To show that Ru-BP does not occur in the cell extract of E. coli wildtype, we performed the assay with just the cell extract of the wildtype without addition of Ru-BP. As a control, we did a second assay containing the cell extract and Ru-BP was added. The samples were taken after 30 min, to demonstrate, that there is no unspecific degradation of Ru-BP. The results are shown in Figure 5. Interestingly there is a small peak found in both assays that corresponds to 3-PGA. No significant increase of the 3-PGA peak was observed in the cell extract when Ru-BP was added. While a number of cellular reactions yields 3-PGA, thus explaining the small amount of 3-PGA in both cell extracts, conversion of Ru-BP to 3-PGA is a specific reaction catalyzed by RuBisCO. This nicely confirmed by the lack of 3-PGA accumulation in the assay containing Ru-BP.

Figure 5: HPLC measurement of cell extract from E. coli KRX wildtype. In the first measurement, ribulose-1,5-bisphosphate was added. The second measurement was performed with the cell extract without adding ribulose-1,5-bisphosphate. Samples were taken after 30 min.

In the third experiment we performed the assay with the cell extract of the wildtype and KRX carrying the construct P_T7 cbbLS_H.neap. taking samples every 5 min to demonstrate the time response of the reaction. Two biological replicates each were performed, which showed both the same results. In Figure 6 the course of the reaction is shown.

Figure 6: RuBisCO activity assay. The cell extract from E. coli KRX carrying P_tac cbbLS_H.neap. and E. coli KRX wildtype was examined for RuBisCO activity. The substrate for the RuBisCO, ribulose-1,5-bisphosphate was added and the time curve of ribulose-1,5-bisphosphate and the product of the enzymatic reaction, 3-phosphoglycerate, was measured via HPLC in 5 minutes intervals.

In the assay containing the wildtype cell extract the peak for Ru-BP remains practically constant over time. In contrast, in the assay containing the cell extract of KRX P_T7 cbbLS_H.neap. shows a clearly decrease of the substrate Ru-BP and a increase of 3-phosphoglycerate over time (Figure 6). It appears that the reaction is not completed after 15 min because there is still some Ru-BP detectable. This may be due to the low reaction rate and high Km of RuBisCO (Sage, 2002).

Conclusion

It can be concluded, that the RuBisCO is active, showing the highly specific conversion of Ru-BP to 3-PGA over time course. Therefore, we successfully proved the activity of the RuBisCO.

References

Andersson, 2008. Catalysis and regulation in Rubisco. Journal of Experimental Botany, vol. 59, pp. 1555-1568
Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. Proceedings of the National Academy of Sciences 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
Jordan, Ogren 1981. Species variation in the specifity of ribulose biphosphate carboxylase/oxygenase. Nature, vol. 291, pp. 513-515
Mann, 1999. Genetic Engineers Aim to Soup up Crop Photosynthesis. Science, vol. 283, pp. 314-316
Parikh et al., 2006. Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E. coli. Protein Engineering, Design & Selection, vol. 19, pp. 113-119
Rosenthal et al., 2011. Overexpressing the C(3) photosynthesis cycle enzyme sedoheptulose 1,7-bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE). BMC Plant Biol., vol. 11, pp. 123
Spreitzer, Salvucci, 2002. RUBISCO: Structure, Regulatory Interactions, and Possibilities for a Better Enzyme. Annu. Rev. Plant Biol., vol. 53, pp. 449-475
Stolzenberger et al., 2013. Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphate from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus. Journal of Bacteriology, Vol. 195, pp. 5112-5122
Hügler, Sievert, 2011. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. Annual Review of Marine Science Vol. 3, pp. 261-289
Sage, Rowan F. „Variation in the K(cat) of Rubisco in C(3) and C(4) Plants and Some Implications for Photosynthetic Performance at High and Low Temperature“. Journal of Experimental Botany 53, no. 369 (2002): 609–20.
Michelet, L. et al., 2013.Redox regulation of the Calvin-Benson cycle: something old, something new. Front Plant Sci, vol. 4.

@@ Line 29: / Line 29: @@
 <span class='h3bb'>'''Sequence and Features'''</span>
 <partinfo>BBa_K1465202 SequenceAndFeatures</partinfo>
+<html>
+<h2>Overview</h2>
+For the verification of RuBisCo expression, we analyzed protein expression of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> KRX containing the construct P<sub>T7</sub><i>cbbLS<sub>H. neap. </sub></i> and as a second verification, protein expression using the construct <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1465213" target="_blank">BBa_K1465213</a>. Cultivations were carried out as described in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">Cultivation for Expression of recombinant proteins</a>. <br><br>
+<h2>SDS-Pages</h2>
+Samples were generated using the protocol for <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">Fast Cell Lysis for SDS-PAGE. </a> The results are shown in Figure 2 and 3.
+<p>
+<center>
+<div class="element" style="width:450px">
+    <a href="https://static.igem.org/mediawiki/2014/8/8e/Bielefeld-CeBiTec_14-10-16_T7_Hneap_Rubisco.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/8/8e/Bielefeld-CeBiTec_14-10-16_T7_Hneap_Rubisco.jpg" width="450px" align="center"></a><br>
+<font size="2" style=""><b>Figure 2:</b> Protein expression of RuBisCO<sub><i>H. neap.</i></sub> over time by <i>E. coli</i> KRX carrying P<sub>T7</sub><i>cbbLS<sub>H. neap. </sub></i> after induction with 0.1 % rhamnose.</font>
+</div>
+</center>
+<br>
+<center>
+<div class="element" style="width:450px">
+    <a href="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_ptac_Hneap_Rubisco.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_ptac_Hneap_Rubisco.jpg" width="450px" align="center"></a><br>
+<font size="2" style=""><b>Figure 3:</b> Protein expression over time of RuBisCO<sub><i>H. neap.</i></sub> by <i>E. coli</i> carrying <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1465213" target="_blank">BBa_K1465213</a> after induction with 0.5 mM IPTG.</font>
+</center>
+<br>
+In both SDS-PAGEs there is a clearly increasing band over the duration of the cultivation. Analysis with MALDI-TOF proved that the band corresponding to a size of a little less than 55 kDa is the large subunit of RuBisCO from <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus" target="_blank"><i>Halothiobacillus neapolitanus</i></a>. The analysis was done via tryptic digestion and an <i>in silico</i> comparison of the measured peptide masses to the predicted  peptide masses. For RuBisCO expressed under control of the T7 promoter seven matching peptide masses were found, the sequence coverage was 15.2 % (MS) and 15.2 % (MS/MS). For RuBisCO expressed via the P<sub><i>tac</i></sub> promoter six matching peptide masses were found and the sequence coverage was 13.5 % (MS) and 13.5 % (MS/MS).
+The small unit of the RuBisCO could only be identified via MALDI-TOF in the samples expressed under the control of the T7 promoter. The analysis was performed as described above, showing three matching peptide masses and a sequence coverage of 35.5 % (MS) and 12.7 (MS/MS). The reason for the problem in identifying the small subunit stems from its small size of 12.8 kDa, making it hard to find in the SDS-PAGE. Still, these results correspond to the verification of protein expression from the plasmid <a href="http://www.addgene.org/52065/"><i>pHnCBS1D</i></a> which we used as the basis for purifying of carboxysomes. Expression of this plasmid gave only in one of three experiments the proof of expression the small subunit.
+<br>
+<br>
+<h2>In vitro assay</h2>
+For the verification of RuBisCO activity, we performed an <i>in vitro</i> assay measuring variances for ribulose-1,5-bisphosphate and 3-phosphoglycerate, substrate and product of the RuBisCo, in the cell extract of KRX wildtype and KRX carrying the construct P<sub>T7</sub> <i>cbbLS</i><sub><i>H.neap.</i></sub>. The method for the <i>in vitro</i> assay is described in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RuBisCOactivityassay" target="_blank">RuBisCO activity assay</a> and the measuring via HPLC in the protocol for <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HPLC" target="_blank">HPLC. </a> <br>
+The first measurement with HPLC was made to identify substrate and product of the RuBisCO, Ru-BP and 3-PGA. Therefore, standards containing just one of the substances were measured (Figure 4). The substances are clearly separable with a retention time of 14.4 min for Ru-BP and 12,6 min for 3-PGA.
+<center>
+<div class="element" style="width:700px">
+    <a href="https://static.igem.org/mediawiki/2014/0/0f/Bielefeld-CeBiTec_14-10-17_rubisco_assay_3.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/0f/Bielefeld-CeBiTec_14-10-17_rubisco_assay_3.png" width="700px" align="center"></a><br>
+<font size="2" style=""><b>Figure 4:</b> Standards for the RuBisCO activity assay. The first measurement was ribulose-1,5-bisphosphate, the second was 3-phosphoglycerate.</font>
+</div>
+</center>
+<br>
+To show that Ru-BP does not occur in the cell extract of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> wildtype, we performed the assay with just the cell extract of the wildtype without addition of Ru-BP. As a control, we did a second assay containing the cell extract and Ru-BP was added. The samples were taken after 30 min, to demonstrate, that there is no unspecific degradation of Ru-BP. The results are shown in Figure 5. Interestingly there is a small peak found in both assays that corresponds to 3-PGA. No significant increase of the 3-PGA peak was observed in the cell extract when Ru-BP was added. While a number of cellular reactions yields 3-PGA, thus explaining the small amount of 3-PGA in both cell extracts, conversion of Ru-BP to 3-PGA is a specific reaction catalyzed by RuBisCO. This nicely confirmed by the lack of 3-PGA accumulation in the assay containing Ru-BP.
+<br>
+<center>
+<div class="element" style="width:700px">
+    <a href="https://static.igem.org/mediawiki/2014/f/f0/Bielefeld-CeBiTec_14-10-17_rubisco_assay_2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/f/f0/Bielefeld-CeBiTec_14-10-17_rubisco_assay_2.png" width="700px" align="center"></a><br>
+<font size="2" style=""><b>Figure 5:</b> HPLC measurement of cell extract from <i> E. coli </i> KRX wildtype. In the first measurement, ribulose-1,5-bisphosphate was added. The second measurement was performed with the cell extract without adding ribulose-1,5-bisphosphate. Samples were taken after 30 min.</font>
+</div>
+</center>
+<br>
+In the third experiment we performed the assay with the cell extract of the wildtype and KRX carrying the construct P<sub>T7</sub> <i>cbbLS</i><sub><i>H.neap.</i></sub> taking samples every 5 min to demonstrate the time response of the reaction. Two biological replicates each were performed, which showed both the same results. In Figure 6 the course of the reaction is shown.
+<br>
+<center>
+<div class="element" style="width:800px">
+    <a href="https://static.igem.org/mediawiki/2014/4/49/Bielefeld-CeBiTec_14-10-17_rubisco_assay_1.png
+" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/49/Bielefeld-CeBiTec_14-10-17_rubisco_assay_1.png
+" width="800px" align="center"></a><br>
+<font size="2" style=""><b>Figure 6:</b> RuBisCO activity assay. The cell extract from <i> E. coli </i> KRX carrying P<sub><i>tac</i></sub> <i>cbbLS</i><sub><i>H.neap.</i></sub> and <i> E. coli </i> KRX wildtype was examined for RuBisCO activity. The substrate for the RuBisCO, ribulose-1,5-bisphosphate was added and the time curve of ribulose-1,5-bisphosphate and the product of the enzymatic reaction, 3-phosphoglycerate, was measured via HPLC in 5 minutes intervals.</font>
+</div>
+</center>
+<br>
+In the assay containing the wildtype cell extract the peak for Ru-BP remains practically constant over time. In contrast, in the assay containing the cell extract of KRX P<sub>T7</sub> <i>cbbLS</i><sub><i>H.neap.</i></sub> shows a clearly decrease of the substrate Ru-BP and a increase of 3-phosphoglycerate over time (Figure 6). It appears that the reaction is not completed after 15 min because there is still some Ru-BP detectable. This may be due to the low reaction rate and high Km of RuBisCO (Sage, 2002). <br><br>
+<h2>Conclusion</h2>
+It can be concluded, that the RuBisCO is active, showing the highly specific conversion of Ru-BP to 3-PGA over time course. Therefore, we successfully proved the activity of the RuBisCO.
+<br><br>
+<h2>References</h2>
+  <p>
+    <ul>
+       <li id="andersson2008">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+        Andersson, 2008. Catalysis and regulation in Rubisco. <a href="http://jxb.oxfordjournals.org/content/59/7/1555.full.pdf">Journal of Experimental Botany</a>, vol. 59, pp. 1555-1568
+  </div>
+</div>
+</li>
+        <li id="bonacchi2012">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+Bonacci, Walter, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, und David F. Savage. „Modularity of a Carbon-Fixing Protein Organelle“. <a href="http://www.ncbi.nlm.nih.gov/pubmed/22184212" target="_blank">Proceedings of the National Academy of Sciences</a> 109, Nr. 2 (1. Oktober 2012): 478–83. doi:10.1073/pnas.1108557109.
+  </div>
+</div>
+</li>
+       <li id="jordan1981">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+        Jordan, Ogren 1981. Species variation in the specifity of ribulose biphosphate carboxylase/oxygenase. <a href="http://www.nature.com/nature/journal/v291/n5815/pdf/291513a0.pdf">Nature</a>, vol. 291, pp. 513-515
+  </div>
+</div>
+</li>
+<li id="mann1999">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+       Mann, 1999. Genetic Engineers Aim to Soup up Crop Photosynthesis. <a href="http://www.sciencemag.org/content/283/5400/314.full" target="_blank">Science</a>, vol. 283, pp. 314-316
+  </div>
+</div>
+</li>
+<li id="parikh2006">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+       Parikh et al., 2006. Directed evolution of RuBisCO hypermorphs through genetic selection in engineered <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a>. <a href="http://peds.oxfordjournals.org/content/19/3/113.long" target="_blank">Protein Engineering, Design & Selection</a>, vol. 19, pp. 113-119
+  </div>
+</div>
+</li>
+<li id="rosenthal2011">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+       Rosenthal et al., 2011. Overexpressing the C(3) photosynthesis cycle enzyme sedoheptulose 1,7-bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE). <a href="http://www.biomedcentral.com/1471-2229/11/123" target="_blank">BMC Plant Biol.</a>, vol. 11, pp. 123
+  </div>
+</div>
+</li>
+<li id="spreitzer2002">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+       Spreitzer, Salvucci, 2002. RUBISCO: Structure, Regulatory Interactions, and Possibilities for a Better Enzyme. <a href="http://www.annualreviews.org/doi/full/10.1146/annurev.arplant.53.100301.135233" target="_blank">Annu. Rev. Plant Biol.</a>, vol. 53, pp. 449-475
+  </div>
+</div>
+</li>
+<li id="stolzenberger2013">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+       Stolzenberger et al., 2013. Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphate from the Facultative Ribulose Monophosphate Cycle Methylotroph <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.methanolicus" target="_blank"><i>Bacillus methanolicus</i></a>. <a href="http://jb.asm.org/content/195/22/5112.long" target="_blank">Journal of Bacteriology</a>, Vol. 195, pp. 5112-5122
+  </div>
+</div>
+</li>
+<li id="huegler2011">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
+  <div id="text">
+Hügler, Sievert, 2011. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. <a href="http://www.ncbi.nlm.nih.gov/pubmed/21329206">Annual Review of Marine Science</a> Vol. 3, pp. 261-289
+  </div>
+</div>
+</li>
+<li id="sage2002">
+<div class="element" style="margin_10px 10px 10px 10px; padding:10px 10px 10px 10px">
+ <div id="text">
+Sage, Rowan F. „Variation in the K(cat) of Rubisco in C(3) and C(4) Plants and Some Implications for Photosynthetic Performance at High and Low Temperature“. <a href="http://www.ncbi.nlm.nih.gov/pubmed/11886880" target="_blank">Journal of Experimental Botany</a> 53, no. 369 (2002): 609–20.
+</div>
+</div>
+</li>
+<li id="michelet2013">
+<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
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+Michelet, L. et al., 2013.Redox regulation of the Calvin-Benson cycle: something old, something new. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838966/">Front Plant Sci</a>, vol. 4.
+  </div>
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