Gold Medal 2 Special Awards: Best New Basic (Gene) Part, and Best Applied Design

1. Introduction
Sugar overdoses not only make you overweight but cause a serious health problem such as obesity, type II
diabetes, atherosclerosis and Alzheimer's disease, etc.
Keep Body in Shape
You can do more exercise, or choose artificial sweeteners as alternatives, or extremely take medicines
(Stony_Brook, 2015), or reduce appetite (NTU-LIHPAO-Taiwan, 2015). However, the limited appetite will take you
away from sweet happiness. Sweeteners or glucose intervention by drugs has serious side effects.
Life of Sugar in the Body
The sugar enters your mouth and binds to the sweet receptors in taste buds inducing a pleasure signal in the brain
through the dopamine reward system. Next, the glucose goes to digestive system, where glucose will be absorbed
by the small intestine and enter bloodstream. However, the excess of blood glucose will disturb homeostasis and
harm your body.
Methodology
Experiment & Result
Conclusion
Glucose responsive suicide circuit
To clean the engineered bacteria after running out of glucose, we combined a glucose responsive element and a
repressor system to regulate suicide circuit by improving and extending the functions of the existing BioBrick parts
of a glucose responsive promoter Pcar [BBa_K861171] (WHU-China, 2012), PhlF repressor system [BBa_K1725041,
BBa_K1725001] (Glasgow, 2015), Lysis gene [BBa_K117000] (NTU-Singapore, 2008), and nuclease NucA
[BBa_K1159105] (TU-Munich, 2013)
Glucose Transporter
To enjoy sugar and prevent glycemic burden, we hijacked the
glucose before entering the intestinal cells by genetically engineering
probiotics to express transporters with high efficiency of glucose
absorption. The glucose transporter of Salmonella has lower Km
(Table 1) and higher affinity, which was cloned and driven under a
constitutive promoter CP29. Table 1. Km values of glucose transporters from organisms
Procedure and Materials
The bacteria were cultured in LB with 34
μg/ml of Cm at 37°C overnight. The next day,
OD600 was adjusted in M9 minimal media with
glucose. After 4 hours, GFP (488/518nm) and
OD600 were measured with a microplate reader
(BioTek). The culture media diluted 106 times
were spreading onto the plate and the colonies
were counted on the 3rd day. Glucose level was
analyzed with Glucose Assay Kit (Sigma)
Fig 2. Glucose assay in different concentrations of glucose
Glucose Absorption Analysis
In Fig. 3, cell growth of E. coli
expressing the Na+/Glu transporter
was slightly higher than the control
group. The glucose began to be
absorbed at the 3rd hour. The glucose
uptake efficiency was achieved up to
97% in Na+/Glu group and greater
than in control group with 1.2 times
difference.
Fig 3. Cell growth and glucose uptake
efficiency at different time point
Suicide Circuit Assay
In Fig. 4, the OD of E. coli with lysis and nuclease genes
(rSuicide) was gradually reduced to 1.89 much less than
average 2.71 in control group (rGFP). In Fig. 5, the survival
rate and cell numbers was decreased to 34% and 671
compared to the control 56% and 1120, respectively, indicating
that killing process was triggered in the loss of glucose.
Fig 4. E. coli growth with (rSuicide)
or without (rGFP) suicide genes in
response to glucose
Fig 5. The cell numbers and viability of
E. coli with (rSuicide) or without (rGFP)
suicide genes in response to glucose
Lactobacillus recombination vector
To stably engineer Lactobacillus as a food supplement by chromosomal
homologous recombination, we chose a region downstream of slpA gene
(Fig. 1), which encodes a surface-layer protein with a strong constitutive
promoter activity (Appl Environ Microbiol., 2011).
Fig 1. The schematic diagram of gene integration location
The probiotic recombination vector
The part CP29-RBS-aeBlue [BBa_K1033280]
with E-X-S-P sites was inserted to make the
vector suitable for standard BioBrick assembly.
RBS-EmR-CP29-RBS-aeBlue/pLBA169
We created a probiotic recombination vector compatible with standard BioBrick assembly. We also enhanced
the glucose absorption efficiency with 1.2 times by the engineered E. coli. Further, we extended the functions
of existing parts by connecting glucose responsive promoter, repressor system, lysis and nuclease gene.