Team:LINKS China/Contribution
Overview
This year LINKS_China aims to produce a leather substitute of bacterial cellulose membranes grown from SCOBY and later bound with layers of spider silk fibroin forming a net to improve its tensile properties. While, also using E.coli to dye our synthetic leather from the natural pigments produced, creating our final product.
We have achieved the following contributions below:
Method of Bacterial Cellulose Membrane
Firstly, we isolated four different colonies of Komagataeibacters from Kombucha tea which includes: the K.intermedius strain 12, 25, 40 and B2. We tried and succeeded in optimizing the efficiency of the production of bacteria cellulase membrane pathway. Finally producing a leather substitute that improves the traditional leather industry
We produced a systematic method when testing the growth for the most productive strain in a 15ml and 50ml centrifuge tube, 2L glass bowl and 4L glass tank respectively. We have provided the method of bacteria cellulose membrane culture and the cleaning process for it in our protocol section.
LINK: https://2021.igem.org/Team:LINKS_China/Experiments
Figure 1. BCM in different culture environments. A) Komagataeibacter sp. in 14mL culturing tube with 5mL YPD after shake culturing. B) Komagataeibacter sp. in 50mL centrifuge tube with 15mL YPD. C) SCOBY in 2L glass bowl with 600mL YPD. D) Raw BCM grown from SCOBY in 4L glass tank with 2L YPD. E) Raw BCM from SCOBY in 4L glass tank with 2L YPD curled up in hand. Growth medium for all is YPD + 0.5% glucose. Aerobic conditions are ensured for all cultures.
Spider Silk Modification of BCM’s properties
We used the spider silk component BBa_K3264000 to directly express spider silk fibroin. We purified and determined the concentration of our spider silk protein. Later layering bacteria cellulose membrane with a spider silk fibroin net forming hydrogen bonds with other fibroins in the membrane. In order to affirm the bacteria cellulose membrane’s properties and potential uses, we performed experiments testing the production rate, softness and tensility respectively. When adding the protein expressed by the spider silk components, the stress and softness results are shown to be more outstanding compared to when it is not added.
Furthermore, we used BBa_K3264000 as our base material to design and construct an improved bio-brick BBa_K4011008 our CBM-2REP. It’s improved effect is better as it binds more efficiently to the bacteria cellulose membrane.
Spider Silk Modification of BCM’s properties
LINK: https://2021.igem.org/Team:LINKS_China/Engineering
Figure 2. Expression and purification of 2Rep and CBM3-2Rep-CBM3. A) Schematic representation of 2Rep and CBM3-2Rep-CBM3 in E. coli BL21(DE3). B) SDS-PAGE analysis of 2Rep and CBM3-2Rep-CBM3 expression. C) SDS-PAGE analysis of his-tag purification of CBM3-2Rep-CBM3 and CBM2-2Rep-CBM2. D) BCA test of 2Rep and CBM3-2Rep-CBM3. The blue dot represents 2Rep while the pink dot represents CBM3-2Rep-CBM3.
Improvement of Pigment Bio-brick
We used the Indigo expressing pathway BBa_K3264022 and used the standardized samples of Trp, 6-Cl-Trp and 6-Br-Trp to produce Tyrian red and Tyrian purple. Then used halogenated samples of them to produce a natural pigment of Tyrian purple, and measured the expressed pigment while making a series of characterizations to its yield becoming BBa_K4011004-BBa_K4011005.
Figure 3- Characterisation of BBa_K3264022 and BBa_K4011004
Expression of the Yeast Toolkit
Throughout the year we have worked closely with AISSU on expressing the yeast toolkit together by sharing the optimal condition guidelines, and so verified the construction of the plasmid and its expression in yeasts. After several unsuccessful trials, team AISSU-Union discovered the optimal assembly condition for the Yeast Toolkit, and they test the expressed DNA with colony PCR to prove a successful expression.
LINK:https://2021.igem.org/Team:LINKS_China/Partnership
NeoLeathic Tanner
We have modified a NeoLeathic Tanner for our project, a steel box that is capable of moderating constant temperatures in order to provide an optimum environment for the development of any bacterial cellulase in the future, yet being sterile and bacteria-free. We believe that it is a helpful reference for any future static fermentation.
LINK:https://2021.igem.org/Team:LINKS_China/Hardware
Standardized Pigment Detection Method
We have standardized the detection experiment for pigment, this systematic method is capable in helping any future teams who includes the production of any pigment materials.
LINK: https://2021.igem.org/Team:LINKS_China/Measurement
