Team:Stuttgart/Improve
Project
Improvement of biobricks
The biobrick we wanted to improve was BBa_I746916 containing genetic information for superfold green
fluorescent protein (sfGFP). As BBa_E0040 (GFP) has a similar function but already being optimized, we
wanted to further optimize this biobrick the same way.
Therefore, our goal was to generate a codon-optimized plasmid containing genetic information for sfGFP
or GFP for the production in vibrio natriegens with the aim to compare growth and fluorescence
of transformed vibrio natriegens with codon optimized and non-optimized sfGFP/GFP.
Two methods were chosen: an improvement via mutagenesis-PCR and improvements via synthesized sfGFP and
GFP. All enzyme reactions and kits were performed according to the provided protocol by Zymo Research
and NEB.
Improvement via PCR
Biobricks BBa_E0040 (GFP) and BBa_I746916 (sfGFP) were used from iGEM DNA Distribution Kit Plates and transformed into electrocompetent DH5α via electroporation (Protocol_Transformation.pdf).
Clones were picked and DNA was extracted via ZR Plasmid Miniprep-Classic Kit (Protocol_Plasmid_Preparation.pdf). PCR was performed introducing one mutation using the primers listed in Table 1.
|
Position |
Mutation |
|---|---|
| sfGFP 84 | TCC/TCT |
| sfGFP 210 | TGC/TGT |
| GFP 219 | AGA/AAA |
| GFP 144 | TGC/TGT |
PCR product was digested with DPN1. Agarose-gel extraction (Protocol_Gel_Extraction.pdf) was performed with the kit Zymoclean Gel DNA Recovery Kit and ligation was conducted with KLD enzyme mix (Protocol_BioBrick_Cloning.pdf).
DNA was transformed into electrocompetent DH5α via electroporation (Protocol_Transformation.pdf). In total, two mutagenesis steps were performed for sfGFP and GFP.
Improvement via synthesized inserts
Inserts for the backbone pSB1C3 were synthesized with the following exchanges:
|
Position |
Mutation |
|---|---|
| sfGFP 84 | TCC/TCT |
| sfGFP 258 | TCC/TCT |
| sfGFP 297 | TCC/TCT |
| sfGFP 210 | TGC/TGT |
| GFP 219 | AGA/AAA |
| GFP 288 | AGA/AAA |
| GFP 366 | AGA/AAA |
| GFP 504 | AGA/AAA |
| GFP 645 | AGA/AAA |
| GFP 144 | TGC/TGT |
| GFP 210 | TGC/TGT |
| GFP 606 | TCC/TCT |
Designed inserts and vector backbone (pSB1C3) were amplified via PCR, digested with EcoRI and PstI and the backbone was dephosphorylated using Antarctic Phosphatase afterwards (Protocol_BioBrick_Cloning.pdf). The samples were loaded onto an agarose gel (Protocol_Agarose_Gel.pdf) and the corresponding bands were extracted (Protocol_Gel_Extraction.pdf). For ligation, T4 DNA ligase was used. The product was transformed into vibrio natriegens (Protocol_Transformation.pdf).
As a result, transformation was not possible into vibrio natriegens. Several methods were tested including various recovery media and voltages for electroporation. Also, new buffers and enzymes were tested for cloning and ligation to exclude mistakes in biobrick formation. Other methods for transformation including chemocompetent cell production and transformation as well as mating will be tested to increase transformation rate of vibrio natriegens.