Introduction

As already mentioned in the project section we decided to work with the Calvin-cycle. There are different reasons for this. One the one side we searched for a method to cultivate aerobically and on the other side we searched for a cycling system which is not given in every carbon dioxide fixation possibility.
In E.coli are three enzymes missing to enable the whole cycle. Our goal is to transform the missing genes from different organisms. Our main research is based on a publication by Bonacci et al., 2011. The idea was to use the carboxysom as a microcompartiment for E.coli from Halothiobacillus neapolitanus for an efficient RuBisCO activity. The problem of the RuBisCO is the affinity to oxygen which lead to non fixating cycle. A carboxysom establishs a higher concentration of carbon dioxide in the microcompartiment. The substrate for the RuBisCO is provided by an enzyme called phosphoribulokinase which took from Synechococcus elongatus. The last missing enzyme was taken from Bacillus methanolicus and is called sedoheptulose 1,7-bisphosphatase (Stolzenberger et al., 2013).
A problem for our system would be the glycolysis which we aimed to inhibit with a knock-down of the phosphofructo kinase. We called this system the gluco-switch.
The main goal for characterization was to establish a new enzyme purification system based on an intein tag with chitin binding domain. It could be used by integrating a coding sequence into the vector.

Phosphoribulokinase

The sequence of the phosphoribulokinase was synthesized to remove illegal restriction sites and to optimize the codon usage for E.coli. We were able to transform the prk into E.coli but without ribosom binding site. As a template for the synthesis we used the prkA of Synechococcus elongatus.
The toxicity of the prk in E.coli was described previously by Parikh et al., 2006 and Bonacci et al., 2012. The toxicity results through the accumulation of ribulose 1,5-bisphosphate which can not be further metabolized. We performed an enzyme assay to identify the functionality of the prkA in E. coli. For this we cultivated the strain and made crude cell extract. The cell extract was incubated for 1 h at 37°C. We compared different approaches. First we incubated a wildtype strain, secondly we incubated the crude cell extract and thirdly we incubated the crude cell extract with 1 mM ribulose 5-phosphate which is the substrate for the prk. The target was to identify ribulose 1,5-bisphosphate with the HPLC. Because the wildtype is not able to produce ribulose 1,5-bisphosphate the prk activity should be easily seen. We were not able to identify the product with HPLC in all approaches. For performing a SDS-Page we cultivated the prkA and induced with 1 mM IPTG. In comparison to the wildtype the prkA carrying strain showed a similar growth behaviour. The resulting SDS-Page is shown below.

Toxicity of phosphoribulokinase without RuBisCO

The prkA should have a molecular size of 38 kD. There is a clear band shown between 35 kD and 40 kD. This fragment was cut out to use it for a MALDI-TOF to identify the protein. With the MALDI-TOF we were able to identify three peptides of the prkA which is sufficient.
The result of our overall approaches looked like the prkA is expressed by E.coli but does not show a functionality in an in vitro assay. A possible problem is the light dependent activation of the prk. This activation is triggered by thioredoxin which is maybe not present sufficiently. As previously described it would be possible to activate the prkA by adding DTT in the enzyme assay (Hariharan et al., 1998) which would be our next try for an enzyme assay.

Sedoheptulose 1,7-bisphosphatase

For the characterization of the sedoheptulose 1,7-bisphosphatase (SBPase / glpX) we did an enzyme assay with a His-Tag purification as described before (Stolzenberger et al., 2013).
The proteins were overexpressed by adding 1 mM IPTG for the T7 promotor. The increasing amount of protein could be verified through a SDS-PAGE..

Proteinexpression of fba

Proteinexpression of tkt

Proteinexpression of glpX

All three SDS-Gels showed a clear band like described in Stolzenberger et al., 2013. We purified the transketolase (tkt) and the fructose bisphosphate aldolase (fba) as well as the sedoheptulose 1,7-bisphosphatase with the His-Tag mediated purification system.

Protein purification of fba

Protein purification of tkt

Protein purification of glpX

For the purified enzymes we performed a Bradford assay.

The Bradford assay showed high concentrations of Tkt and Fba as well as a very low concentration of GlpX. After the purification we performed an enzyme assay as shown below.

SBPase assay

Molecule Formula Molecular weight [M-H] Fructose-6-P C6H15O9P 259.021 Glyceraldehyd-3-P C3H7O6P 168.99 Dihydroxyacetone-P C3H7O6P 168.99 Xylulose-5-P C5H11O8P 229.01 Erythrose-4-P C4H9O7P 199.00 Sedoheptulose-7-P C7H15O10P 289.03 Sedoheptulose-1,7-BP C7H16O15P2 368.99

The product of the reaction, sedoheptulose 7-phosphate, could be identified via HPLC. We made different approaches to characterize all reactions.



Reaction mix:

• 20 mM Fructose 6-phosphate
• 20 mM Glyceraldehyde 3-phosphate
• 20 mM Dihydroxyacetonephosphate
• 10 µM Thiamine pyrophosphate
• 2 mM Manganese chloride
• 50 mM Tris-HCl

In the first approach we add no enzyme to verify that no product is generated. The second approach includes the transketolase which does the reaction of F6P and GAP to erythrose 4-phosphate. In the third approach fructose bisphosphate aldolase was added which converts erythrose 4-phosphate with dihydroacetonephosphate to sedoheptulose 1,7-bisphosphate. In the last approach the sedoheptulose 1,7-bisphosphatase (glpX) was added which results in sedoheptulose 7-phosphate. All intermediates could be verified in all approaches as expected. This measurement showed the activity of the SBPase in vitro (Xylulose 5-phosphate is a byproduct of one of the enzymatical reactions).

Comparison of 37°C and 50°C of the in vitro assay (all enzymes).

We did a comparison between 37°C and 50°C. The transketolase and aldolase performed with a higher activity at 37°C which resulted in more products. The sedoheptulose 1,7-bisphosphatase acitvity is higher at 50°C but also shows activity at 37°C. We could identify this behaviour because of the accumulation of sedoheptulose 1,7-bisphosphate in the 37°C approach. The second approach has a lower concentration of this substance but showed a higher concentration of sedoheptulose 7-phosphate which is due to the higher activity of the SBPase at 50°C. This result means that our approach is able to enable the whole Calvin-cycle with glpX in E.coli.
We decided to use glpX as a target for amplification and transformation because of the shown acitivity. The finished construct of ptac_glpX in pSB1C3 was cultivated in M9 glucose in comparison to the wildtype. We performed two biological replicates and two technical replicates.

Cultivation experiment in M9 glucose

The cultivation shows that the modified strain has a longer lag phase before the entrance in exponential phase. Two hours after IPTG induction of the strain results in a decrease of growth in comparison to the uninduced strain. There are two possible explanations for this behaviour. One the one side IPTG acts as a poison for bacteria which may result in growth decrase and on the other side the production of the protein can result in decreased grwoth. We exclude IPTG as a reason because earlier cultivations showed that 1 mM IPTG have no effects on the wildtype growth. Entering the stationary phase takes place one hour before the induced strain. The induced strain also shows a higher OD. By inducing the SBPase in E. coli the substances for glycolysis are deflected towards other pathways. These reactions are reversible which means that the glucose of the M9 medium is not metabolized in another pathway. This time shifted use of glucose results in a higher OD in two biological replicates.
This result shows that the SBPase does not limit the growth maximum of E. coli. The glucose concentration confirmes these results. The wildtype consumes the glucose faster than the mutant strains.

Cultivation experiment in M9 xylose

The cultivation was repeated with xylose because we think of using xylose as an additional carbon source if the calvin cycle has a los efficiency. This second cultivation shows the impact of the induction more clearly. The induced mutant strain has a slower growth rate in comparison to the uninduced mutant and the wildtype. This indicates that the lack of glucose in the medium has a large impact because necessay intermediates for the glycolysis were used by the SBPase.

Gluco-Switch

For the knock-down of the pfkA we used the synthesised sRNA:pfkA. Herewith we wanted to down-regulate the glycolysis to prohibit the bacteria to use sugar as a carbon source for our later experiments. This should cause a reduction of the growth of our bacteria carrying the plasmid. We tried to show this in a cultivation which results are shown in figure XXXX.

Figure XX:

Neither the construct with the p_tac promoter nor the one with the T7 promoter showed a difference between induced and not induced. As we used M9 glucose as medium for the cultivation there should be a difference in the growth. The HPCL results did not give another hint for the functioning of the sRNA because the induced culture as well as the not induced one showed the same use of glucose.
Maybe it was not enough to only take the sRNA of pfkA so another try would be to integrate the sRNA:pfkB downstream of the sRNA:pfkA to be sure to cover the activity of both phosphofructokinases. Additionally the building of the sRNA should be checked.

Protein purification

For the purpose of characterizing our BioBricks we thought of using enzyme assays to verify the functionality of different proteins. Enzyme assays depend on purified enzymes. A typical purification approach is the His-Tag mediated purification system. The disadvantage of this system is that the tag remains attached at the enzyme after the purification and has to be cleaved afterwards. A further development of this system is the intein tag mediated purification.
By adding an intein tag attached to a chitin binding domain to the enzyme of interest a purification can be achieved. The chitin binding domain binds the column on which chitin beads are stored. After adding binding buffers and washing solutions an elution with DTT allows to cut the attachment of the intein tag to the coding sequence. The enzyme is eluted from the column and can be stored in the desired buffer. The chitin binding domain and intein tag can be eluted from the column afterwards to reuse the column.
We implemented this system in the pSB1C3 backbone by combining the T7 promotor with RBS and intein tag with chitin binding domain.

By designing gibson assembly primers with following overhangs it is possible to add a coding sequence between the first and the second part of the purification vector (add the gene specific part behind the overhang with the right orientation):
>GSP_fw
CTATAGGGAAAGAGGAGAAAT
>GSP_rev
CTAGTGCATCTCCCGTGATGCA

Note: The stop codon of the coding sequence has to be deleted through primer design.

It may be possible to redesign the pSB1C3 backbone to the purification vector by including the T7 and RBS as well as the intein tag with chitin binding domain into the backbone. The restriction sites for BioBrick assembly may be placed in between both patterns. This would allow an in frame addition of the coding sequence by using BioBrick assembly (Note again: The stop codon has to be deleted during the amplification of the coding sequence).
Because of problems during the transformation of the coding sequences we were not able to characterize this BioBrick.

References

• 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
• Bonacci et al., 2011. Modularity of carbon-fixing protein organelle. PNAS, vol. 109, pp. 478-483
• 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
• Hariharan et al., 1998. Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae. Plant Physiol. Vol. 117, pp. 321-329