Difference between revisions of "Part:BBa K1983000"
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This result shows that incorporating membrane transporter for phenylalanine was valuable for the whole systems avtivity <i>in vivo</i>. | This result shows that incorporating membrane transporter for phenylalanine was valuable for the whole systems avtivity <i>in vivo</i>. | ||
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=Contribution= | =Contribution= | ||
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<h3>Cloning</h3> | <h3>Cloning</h3> | ||
− | The enzyme AvPAL was | + | The enzyme AvPAL was restriction based cloned into the pET15b-mCherry backbone while cutting out mCherry. We controlled the appearence of AvPAL as well in an SDS-PAGE and western blot to receive reproducible results. |
[[File:T--Heidelberg--AvPAL detection.png|thumbnail|center|500px|<b>Figure 6. AvPAL SDS-PAGE, Ponceau staining and Western Blot]]. </b> | [[File:T--Heidelberg--AvPAL detection.png|thumbnail|center|500px|<b>Figure 6. AvPAL SDS-PAGE, Ponceau staining and Western Blot]]. </b> | ||
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<h3>Assay of enzyme activity</h3> | <h3>Assay of enzyme activity</h3> | ||
− | [[File:T--Heidelberg--absorbance phe measurements.svg|thumbnail|center|500px|<b>Figure 7. Absorbance of Phe and tCa and measurments in vitro]]. </b> | + | [[File:T--Heidelberg--absorbance phe measurements.svg|thumbnail|center|500px|<b>Figure 7. Absorbance of Phe and tCa and measurments <em>in vitro</em> </b> The first three plots represent the absorbance spetrum of (A) phenylalanine (Phe), in (B) trans-cinnamic acid (tCa) and in (C) the merged graphs of (A) and (B).]] |
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+ | <h2>References</h2> | ||
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+ | <b>1.</b> Lovelock, S. L. and N. J. Turner (2014). "Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity." Bioorg Med Chem 22(20): 5555-5557. | ||
+ | <b>2.</b> Cui, J. D., S. R. Jia, et al. (2008). "Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli." Lett Appl Microbiol 46(6): 631-635. | ||
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Revision as of 10:43, 11 October 2021
Codon optimized AvPAL with C-terminal 6XHis-Tag
Overview
Phenylalanine ammonia lyase (PAL) from Anabaena variabilis is an enzyme that catalyzes breaking down L-phenylalanine to ammonia and trans-cinnamic acid. This part was introduced by Vilnius-Lithuania iGEM Team as a functional part used to break down L-phenylalanine in vivo. This is an improved Stanford-Brown 2013 AvPAL biobrick part. We have removed the T7 promoter and replaced the N-terminal FLAG-tag with a more common C-terminal 6xHis-tag. The C-terminal tag location was chosen assuming it would less likely affect the stability and folding of the protein.
Our team improved the characterization of this part by testing the enzyme's activity in vivo. We also have experimentally measured the expression rate and time to reach steady of the protein in Escherichia coli cells, since the kinetic constants of this enzyme are determined [1]. Expression of the 6XHis-tagged protein is easily detectable via Western Blot. We used pBAD expression system for our project and characterization of this part.
Experiments and Results
PAL was cloned into pET and pBAD expression vectors, with the latter showing better expression results. Thus, further experiments were carried out using pBAD vector and the procedures below were described using this vector (see results page for more information).
Cloning
The received sequences were amplified using PAL-Chis FW/RV primers and digested with Esp3I and XhoI. The fragments containing mutant genes were cloned into pBAD expression vector digested with NcoI and XhoI. Transformant colonies were PCR-screened using pBAD-Pro/Term primers and positive pBAD PAL clone plasmids were sequenced prior to further usage.
Expression assays
SDS-PAGE and Western Blot
pBAD PAL expression was tested in E. coli TOP10 strain, showing positive results on SDS-PAGE gel and Western Blot (Fig. 1).
PAL expression rate over time
Additionally, PAL expression was tested over time to measure the time needed for PAL to reach a steady state concentration inside the cells (Fig 2 and 3).
![](/wiki/images/f/f1/T--Vilnius-Lithuania--PAL_expression_rate_gel.png)
PAL activity in vivo results in optimal conditions (see below) can be used in determining the quantities of PAL inside the cells since the kinetic constants of this enzyme are accessible [1]. It was assumed that if surfactants (60% EtOH) eliminated the impermeability [2] of the membrane, the true values of PAL expression inside the bacteria could be calculated from the known kinetic constants. The expression rate over time was normalized by dividing each measured band score by the OD600 at the corresponding time point.
![](/wiki/images/0/0d/T--Vilnius-Lithuania--PAL_expression_rate.png)
This data is later used in our model of the system, since it describes the time for PAL to reach steady state concentration.
PAL characterizaton in vivo
E.coli expressing PAL was tested under laboratory conditions (Fig. 4) to see if the enzyme is working in vivo. Since PAL is expressed inside the cell, L-phenylalanine has to permeate the membrane and diffuse to the interior. To test the effect of the membrane as a mechanical boundary for L-phenylalanine to pass through, surfactants of varying concentrations were also used. This experiment has proven that E.coli expressing PAL can effectively convert L-phenylalanine to tCA. Also, the effect of surfactants showed that the membrane is one of the limiting factors to the efficiency of the probiotic. The use of surfactants was also helpful to identify the quantities of PAL expressed inside the cells since the membrane boundary effect was eliminated.
Since the previous experiments showed that in order for PAL to work more effectively, the membrane ruggae had to be increased, so the next step was to test the cooperation of a constantly expressed PheP (E.coli L-phenylalanine permease) from a composite biobrick part (BBa_K1983014) with PAL (Fig. 5). The role of PheP was to facilitate the diffusion of L-phenylalanine to the cell‘s interior, thus increasing the effectiveness of conversion to tCA.
![](/wiki/images/c/cb/T--Vilnius-Lithuania--PAL_PheP_POC.png)
This result shows that incorporating membrane transporter for phenylalanine was valuable for the whole systems avtivity in vivo.
Contribution
- Group: iGEM Team Heidelberg 2021
- Author: Franziska Giessler, Silja Malkewitz, Marilena Wittmaack
- Summary: The Part BBa K1983000 was used for our project and further characterized by enzyme activity measurements.
Experiments and Results
Cloning
The enzyme AvPAL was restriction based cloned into the pET15b-mCherry backbone while cutting out mCherry. We controlled the appearence of AvPAL as well in an SDS-PAGE and western blot to receive reproducible results.
.
Assay of enzyme activity
References
1. Lovelock, S. L. and N. J. Turner (2014). "Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity." Bioorg Med Chem 22(20): 5555-5557. 2. Cui, J. D., S. R. Jia, et al. (2008). "Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli." Lett Appl Microbiol 46(6): 631-635.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1265
Illegal XhoI site found at 1702 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1483
- 1000COMPATIBLE WITH RFC[1000]