Part:BBa_K3490011
Causing bacteria lysis when the bacteria leak out the contact lenses.
Our first kill switch design used TetR fused with VP16, tet-off system[1] to control the Colicin E7(BBa_K117000) as our biosafety measure. We will put tetracycline analog into the contact lens, this will prevent the VP16 to activate the lysis gene. If the bacteria leave the contact lens, where the environment has low concentration of tetracycline analog, the lysis gene will be activated.(Fig.1)
There are several options when it comes to tetracycline analog that can act on the tetR protein, changing its DNA binding affinity, however, according to Biomol GmbH-Life Science Shop, 50 mg of Anhydrotetracycline costs 54 US dollars[2]. In search of a cheaper alternative, we turned to tetracycline(TC)[3] and Chlortetracycline(CTC)[4], which has been reported to bind to tetR protein and affect its affinity to tetO.
Fig.1. design schematic of the killswitch using tet-off system
After many attempts constructing this plasmid resulted in failure, we turned to gene knockout as our biosafety masure. However, we can still characterize the effect of TC and CTC on TetR using iGEM BioBrick BBa-K611059.(Fig. 2)
Fig.2. iGEM BioBrick BBa-K611059
BBa-K611059 has two open reading frames, a pTet promoter controlled GFP, and pBAD promoter controlled TetR. As we use arabinose to produce TetR to repress pTet, this allowed us to try different tetracycline analogs and observe GFP signal recovery to assess their potency. Since most tetracycline analogs have antimicrobial activity, we test two different ways to compensate for this effect, heat inactivation of tetracycline analogs and co-transform tetracycline resistance plasmid.
We first transformed the plasmid carrying BioBrick BBa-K611059 into BW25113, which cannot metabolize arabinose, and added different concentrations of heat inactivated CTC[5] to see if the GFP signal is restored.
After 12hr of culture with different concentrations of heat-inactivated CTC and 0.0001% of arabinose, we can see as arabinose was added into the culture, the normalized GFP signal slightly decreased, suggesting BBa-K611059 functions correctly. As 20µg/ml heat-inactivated CTC can rescue the GFP signal, and 30µg/ml will result in higher GFP signal. Also, through the OD600 results we can also conclude the heat-inactivated CTC does not affect E. coli growth.
Fig.3. GFP value according to different concentrations of heat inactivated CTC with 0.0001% of arabinose and the OD600 value
Despite showing that heat-inactivated CTC can activate pTet, we were not satisfied with the dynamic range, so we decided to challenge using TC itself, since this operon originated in response to antibiotic resistance. In concerns of TC will be deadly to E. coli itself, also there has not been reported heat-inactivation TC can activate pTet, we co-transformed a TC resistance plasmid, pACYC184, into BW25113 to conduct further experiment.
Fig.4.a. GFP value according to different concentrations of TC with 0.002% of arabinose and the OD600 value
Fig.4.b. GFP value according to different concentrations of TC with 0.0002% of arabinose and the OD600 value
We first tried two different arabinose concentrations, 0.002% and 0.0002%, both concentrations show repress GFP signal. However, bacteria experienced growth retardation in 0.002% of arabinose, we speculate that the high arabinose concentration induces osmotic stress to the bacteria. Also we can see as TC concentration increases, although the GFP signal remains the same, the OD600 decreases. This indicates that TC can induce pTet to produce GFP but suppress bacteria growth. We speculate that pACYC184 can not withstand such high concentration of TC.
From these results, since both TC and heat-inactivated CTC can activate pTet, but TC seems to affect cell growth at higher concentrations although we supplied the bacteria with resistance plasmid, we can conclude that heat-inactivated CTC is a better inducer compared to TC.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 755
Illegal NheI site found at 1068
Illegal NheI site found at 1091 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |