Part:BBa_K3256440
ccdB*
Introduction
The original part is from BBa_P1010. The modified amino acid sequence includes N95T(shown below). And the protein was truncated at 96th amino acid with a stop codon. After these modifications, the toxin retains the function of suppressing the growth of E. coli, and makes the E. coli more stable from being killed easily.
Result
Cloning
We conducted colony PCR to verify that our target gene was correctly cloned into the E. coli BL21 (DE3).
Figure 1:Colony PCR result of toxin genes after cloning into E. coli BL21(DE3) ccdB* BBa_K3256440
Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned five target genes, respectively, into E. coli.
Functional Test
After confirming the cloning of target genes, we tested their function by measuring the O.D. value after IPTG induction when the O.D. value reached 0.3 and compared with the O.D. value of the control. The O.D. values were documented in every five minutes for seven hours in total. All experiments were completed in triplicates. (Figure 2~6)
Figure 2:Growth curve of E. coli BL21(DE3) with 500uM IPTG induction ccdB toxin gene (blue), and control (orange)
Calculating Toxicity of Toxin Genes
We first fit the control’s experiment data to the following equation, dBT/dt=g⋅BT(1−BT/BMax), and we fit the induced data to another equation, dBT/dt=g⋅BT(1−BT/BMax)−Ttoxin⋅BN⋅[toxin]. Next, we compared the two equations to calculate Ttoxin, the toxicity of the toxin gene (Figure 3). In the end, we chose the ydfD gene, which had the most significant toxicity, and the ccdB gene, which had relatively weak toxicity, moving on to the Functional test with mutagens.
Figure 3:Toxicity of different toxin genes. Ttoxin: The toxicity of toxin gene.
Mutagens Bioassay Function Test
According to the toxin gene functional test, with selected ccdB and ydfD for mutagens bioassay. To see whether our design can work as expected, we put the transformed E. coli into a different dose of mutagens (UV light and EtBr), respectively. Since the mutagen might cause growth inhibition, it was difficult to tell how strong the mutation rate caused by the mutagen was only through the raw data of O.D.600. Therefore, we took the maximum O.D value of the controlled group(which represented the none-induced E. coli treated with the same dose of mutagen) as 100%, and the minimum value is 0% to normalize the data, and we called the normalized data growth population percentage. </p>
Functional Test of Mutagenic Bioassay on UV Light
In the environment, UVB(275～320nm) is the most carcinogenic wave band that humans usually contact according to the reference, so we chose it to be our physical mutagen for the functional test of bioassay. In the experiment, we exposed them to the different intensity of UV light for 5 seconds and induced the toxin gene when its O.D reached 0.3. After that, we documented the O.D. of the E. coli every 5 minutes for 7 hours. The results of the two engineered E. coli with ydfD and ccdB, respectively, under different UV light intensity, were shown below (Figure 4).
Figure 4:The Growth Curve of ccdB with Different Intensity of UV Light.
Functional Test of Mutagenic Bioassay on EtBr
We chose EtBr, a chemical compound that causes mutation and usually seen in laboratories to be our chemical mutagen. First, we made them added different concentrations of EtBr for creating different mutagenic strengths and added 500uM IPTG to induce the toxin gene when its O.D reached 0.3. After that, we documented the O.D value of E. coli every 5 minutes for 7 hours. Below was the EtBr mutagenic effect on the two toxin genes.
Figure 5:The Growth Curve of ccdB with Different Dose of EtBr
From the experiment result, we can see that the curve of ydfD has a more significant drop than ccdB after we induced the toxin gene. This phenomenon can be explained, for the sequence of ccdB has been modified (Figure 6). Therefore, its toxicity has been weakened compare with the intact ydfD sequence.
Mutation rate analysis
According to the growth curve of two mutagenic factors with two selected toxin genes respectively for building up mutagenic bioassay, we further converted the raw data above to mutation rate of the mutagen which we called M2, we used the differential equation from modeling to fit the experiment data.
We were first fixed the parameters from the isolated experiments and essays, and then used Matlab to estimate the mutation rate, M2, with the most appropriate curve. Below is the linear regression of M2 and dose of mutagen (Figure 6,7).
Figure 6:Linear correlation of Mutation rate in UV intensity (ccdB)
Figure 7:Linear correlation of Mutation rate in EtBr concentration (ccdB)
We can see that the M2 we calculate was a highly positive correlation with the dose of mutagens. It verified that by using our designed gene circuit and model, the mutagenic bioassay system we constructed is highly credible and can be further used for mutagenic studies for unknown substances. For the detail of how we build up the simulation equation and the verification of the growth curve model, please see the growth curve model.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
Bernard P, Gabant P, Bahassi EM, Couturier M, Université Libre de Bruxelles. Positive-selection vectors using the F plasmid ccdB killer gene." Gene 1994 Oct 11;148(1):71-4. pmid:7926841
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