Part:BBa_K3256008

Plac+ydfD

This composite part contains lac promoter, RBS and ydfD. More detail about the toxin is shown below.

Introduction

ydfD is one part of the Qin defective prophage in E. coli. According to the research, 99.9% of cell lyses within two hours after ydfD start expressing. dicB, upstream gene of ydfD in E. coli, will prevent bacteria from lysing due to the effect of ydfD. Cell division won’t happen due to inhibiting the enzyme that leads to cell division.

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) ydfD BBa_K3256443

  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.
Figure 2:Growth curve of E. coli BL21(DE3) with 500uM IPTG induction YdfD 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.

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 ydfD 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 ydfD 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 (ydfD)

Figure 7:Linear correlation of Mutation rate in EtBr concentration (ydfD)

  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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 280
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Masuda, H., et al. (2016). "ydfD encodes a novel lytic protein in Escherichia coli." FEMS microbiology letters 363(6): fnw039.