Team:NCTU Formosa/Design
Biobrick Design
Figure 1: Biobrick design
Owing to expressing Gloeobacter rhodopsin(GR) well, our biobrick design has four characteristics. First, we express our GR with T7 inducible promoter. Induced by IPTG, we could make sure that GR will only be produced when
we aim to make it locate and fold properly. Second, GFP was linked with GR with an unique linker for visualizing the transmembrane protein expression more conveniently.
This fusion protein forms only when GR has the correct folding structure, or it would become an inclusion body with GFP. Then, 6X His-Tag is used for purification. Last but not least, we use pET32a for better expression.
GFP linker VS. Correct Protein Folding
The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance same as (GGGGS) 4 linker, but it doesn’t have high homologous repeats in DNA coding sequence.[3] Therefore, if GFP expresses
well, we can ensure that GR proteins fold robustly and are fully soluble and functional.[4] Otherwise, if GR does not express well, it would become an inclusion body with GFP. Furthermore, since the linker is functional,
GFP wouldn’t interfere the function of GR.
Figure 2: Harmonized GR with GFP linker (demonstrated by PyMOL)
Modifications of GR for Better Folding and Expression
To improve GR expression and folding, we performed codon harmonization, optimizing codon sequence to better fit our host strain as opposed to its original host by using an algorithm to mimic native codon usage.
Experimental Design
Step 1:
Transmembrane
Protein Expression
Transmembrane
Protein Expression
Step 2:
Functional Test
Functional Test
Step 1:
Transmembrane
Protein Expression
Transmembrane
Protein Expression
Step 2:
Functional Test
Functional Test
More information see Protocol.
Chassis Selection – E .coli Lemo21(DE3)
Lemo21 is the revolution of BL21. It is used for difficult expressed target genes such as transmembrane protein GR, which expression could be limited by the throughput capacity of the Sec translocase, to express better. Sec
translocase is used for recognizing appropriate segments in the direction of transfer as a stop-transfer sequence for polypeptide chain to be threaded across the membrane. With its tunable T7 expression system, if we successfully
tuned it with L-Rhamnose, Sec translocase wouldn't be limited, and we could prevent inclusion bodies from forming.
Expression Condition – Low Temperature
Slower rates of protein production give newly transcribed recombinant proteins time to fold properly. By reducing incubation temperature, we can decrease aggregation, which is favored at higher temperatures due to the temperature
dependence of hydrophobic interactions. We prove that 16 degrees Celsius is optimal for GR expression.
Expression Tuning – L-Rhamnose
In Lemo21, tunable expression is achieved by varying the level of lysozyme (lysY), the natural inhibitor of T7 RNA polymerase. The level of lysozyme is modulated by adding L-Rhamnose to the expression culture at levels from
zero to 2000 µM. So, we tuned the expression of GR with L-Rhamnose.
Expression Visualization – GFP Measurement
As mentioned, GFP is used to indicate protein expression. We measure the fluorescence with spectrometer to confirm expression before the functional tests.
Carbon Source Reduction– M9 mineral medium
Instead of LB broth, we prepared low glucose M9 mineral medium(0.4% glucose) to culture E. Hybrid. With less essential nutrients, we this alternative medium should help demonstrate the versatility of E. Hybrid.
Essential Component for GR – β-carotene
Instead of all-trans retinal, we use β-carotene produced by our E. coli. It causes conformational change and helps energy transfer in GR, and it could extend the light absorbance of GR. Since we want to produce & β-carotene
with E .coli rather than chemical methods, we transformed BBa_K274200 (CrtEBIY) into E .coli Lemo21. After our experiments, we prove that it can be a cheap alternative to all-trans retinal.
Electron Transport Chain Block – Sodium Azide
We use sodium azide to inhibit oxidative phosphorylation, the last enzyme in electron transport chain, thus ATP production in E .coli have been reduced. We hope it not only slows down the growth of wild type E .coli,
but also enhances the function of GR in E. Hybrid.
Figure 3: Sodium azide blocked the electron transport chain.
Protein Expression Enhancement:
psB1K3 BBa_J04450(RFP)
psB1K3 BBa_J04450(RFP)
We incorporate expression vector to prove the light-induced expression system could be functional when it comes to practical usages. psB1K3 BBa_J04450(RFP) is used as a reporter for us to visualize the function of GR.
Reference
- Takefumi, O., et al. (2019). “X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin”
- GL Rosano, C., et al. (2014) “Recombinant protein expression in Escherichia coli: advances and challenges”
- Xiaoying, C., et al. (2013)“Fusion Protein Linkers: Property, Design and Functionality”
- Waldo, B. , et al. (1999) “Rapid protein-folding assay using green fluorescent protein”
- Que, J. , et al. (2019) “Functional Expression of Gloeobacter Rhodopsin in PSI-Less Synechocystis sp. PCC6803”
- Evelina.,A.,et al.(2008)”Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host”