Part:BBa_K4634013
Single Plasmid AND GATE
AND GATE is a composite part to process two different signal. For this particular AND GATE, temperature and arabinose signal are detected. After undergo heat-shock treatment, T7ptag is transcribed. But as the T7ptag mRNA consist amber stop codons, it cannot be translated to fully functional protein. Meanwhile, if arabinose is added to the system, supD tRNA is transcribed. In the presence of supD, T7ptag mRNA can be translated to a functional T7 RNA polymerase. Hence the mCherry gene controlled by T7 promoter is expressed and fluorescent signal is detectable. This single plasmid AND gate construct has the potential to be tailored to specific needs and reduce the possibility of plasmid loss.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2502
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 1245
Illegal AgeI site found at 2261 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1248
Illegal SapI site found at 961
Biology and usage
This composite part is an AND gate system constructed in one single plasmid. Initially designed to validate the AND gate framework in our overall project plan, it ultimately evolved into an adaptable AND gate module with input and output sections that can be tailored to specific requirements. The input signals for this specific system are arabinose and heat-shock, which are detected by ParaBAD and temperature-sensitive promoter PL-PR, respectively. The downstream reporter gene mCherry is expressed only when both signals are present. This AND gate system employs the mechanism of premature translation termination induced by amber stop codons. When one signal (heat shock) is detected, the downstream gene T7ptag is transcribed into mRNA under the control of the corresponding promoter. However, the T7ptag mRNA contains two amber stop codons, rendering it unable to be translated into a functional T7 RNA polymerase. Meanwhile, if the other signal (arabinose) is also detected, the downstream gene supD is expressed into tRNA supD under the control of the corresponding promoter. tRNA supD has a complementary anticodon to the amber stop codon and can recognize the amber stop codon as serine. Hence, functional T7 RNA polymerase is expressed, which can initiate the output expression under the control of the T7 promoter. The logical function of the AND gate is thus achieved.
In our construction, PL-PR promoters are positioned upstream of T7ptag while arabinose promoter is located upstream of supD. After detecting a certain concentration of arabinose and undergoing heat-shock, the AND gate system will gradually start the expression of the reporter gene mCherry and fluorescence signal can be detected. The successful construction of this composite parts provides a universal monocistronic plasmid AND gate solution. By standard molecular biology techniques, the promoters at the input end can be replaced with other signal detection promoters, and the reporter gene mCherry at the output end can also be replaced with target output genes.
In the construction process of this AND gate system, we paid special attention to the construction of the terminators. Terminators are often ignored in general plasmid construction because prokaryotes utilize two types of transcription termination mechanisms, one of which does not depend on the presence of a terminator. Generally, when using plasmids to express target proteins, we only consider whether the target protein can be expressed normally, without considering whether other downstream genes may leak. However, in the construction of the AND gate, to avoid significant output leakage or input interference caused by promoter penetration, the construction of the terminators is particularly important. In the construction process of the plasmid AND gate system, we added terminators at proper sites through non-traditional PCR techniques. The three promoters (two for input and one for output) and their corresponding terminators divide the entire composite parts into three sections: input 1, input 2, and output. This construction is also beneficial for the construction of complex output. Within any section, multiple gene serial sequences can be inserted between the promoter and the terminator to achieve different purposes.
Components of the construct
(listed in sequence order)
Input 1
- Inducible pBad/araC promoter (BBa_I0500): to detect arabinose signal and be activated when arabinose is present.
- supD( BBa_K4634015): The tRNA with anticodon CUA transporting Serine
- rrnB T1+T2 terminator ( BBa_K3331007)：transcription terminator
Output
- RBS ( BBa_K4634001): Ribosome binding site
- mCherry ( BBa_K4839013): reporter gene, excitation wavelength at 587nm and emission wavelength at 612nm.
- rrnB T1 terminator ( BBa_B0010): transcription termination
T7 promoter ( BBa_I719005BBa_I719005): promoter interacted with T7 RNA polymerase
Input 2
- Temperature-sensitive Promoter（ BBa_K4634017）: to detect heat-shock signal and be activated undergoing heat-shock treatment.
- T7ptag( BBa_K4634016): coding T7 RNA polymerase and containing two amber stop codons.
- rrnB T1+T2 terminator( BBa_K3331007): transcription terminator
Cloning strategy and results
The assembly of all parts in the construction of AND gate followed a recombination strategy. In order to reduce the difficulty of assembling many DNA fragments of different sizes, we have divided the entire cloning process into two steps.
First step
All the primers used in the first step is listed in Table.1.
pBV220-T7ptag-i-f and pBV220-T7ptag-i-f are used for amplifying T7ptag from pJC175e(given by Zhang Shuyi Lab). pBV220-T7ptag-v-f and pBV220-T7ptag-v-f are used for linearizing pBV220 vector (bought from Wuhan Miaoling Biotech). Overhangs are designed according to the sequence to construct proper recombination site at both end of the PCR products. Then PCR products were purified and recombined, forming pBV220-T7ptag. By this step, we ligated T7ptag to the downstream of its input promoter- PL-PR.
pBAD-supD-v-f and pBAD-supD-v-r are used to add supD sequence at the downstream of araBAD promoter in pBAD. supD is only 90bp-long, so a 55bp supD sequence was added to each end of the primers, constructing a 20bp long homologous segment at the end. After homologous recombination, a full-length supD was inserted into the target site. To reduce the chance of self-ligation, pBAD-supD-v-f is paired with pBAD-supD-r while pBAD-supD-v-r is paired with pBAD-supD-f. pBAD vector will be amplified by those two pairs of primers separately, producing two DNA segments with part of supD at one end. Then recombination ligation is performed and pBAD-supD is harvested. By this step, we inserted supD between its input promoter-ParaBAD and rrnB terminators presented in pBAD.
pBV220-T7ptag and pBAD-supD were then send for sequencing to ensure accuracy.
Second step
All the primers used in the second step is listed in Table 2.
pAND-T7-mCherry-r and pAND-T7-mCherry-f are used to amplify T7 promoter-RBS-mCherry using pBV220-mCherry as vector. T7 promoter-RBS sequence is added to pAND-T7-mCherry-f, after PCR reaction, the product would have the promoter and RBS added.
Then pAND-PL-PR-T7ptag-f and pAND-PL-PR-T7ptag-r are used to amplify TcI-PL-PR-T7ptag from pBV220-T7ptag. pAND-araBAD-supD-f & pBAD-supD-r, pAND-araBAD-supD-r & pBAD-supD-f, two pairs of primers are applied separately to linearized pBAD-supD. We specially designed our primers to amplified rrnB terminators in both PCR reactions. Then four PCR products are purified.
To prevent wrongfully ligation during recombination caused by the extra rrnB terminators, we applied another round of overlap PCR reaction. TcI-PL-PR-T7ptag and the product amplified by pAND-araBAD-supD-f & pBAD-supD-r served as vectors and pAND-T7-mCherry-f & pBAD-supD-r served as primers. After PCR reactions, these two segments are combined into a longer segments.
Then In-Fusion system was set up to recombined the three segments. The product was transformed to E.coli DH5α and identified by colony PCR (primers are listed in table 3.). The results show all three monoclonal colony has a single bright band at about 3.4kb(Fig.2). Thus this three monoclonal colony were preliminarily identified. Plasmids were prepared and sent for sequencing for validation.
Hence, pAND was born.
Function validation of pAND
We conducted an experiment to verify the function of pAND. Since the working mechanism of the AND gate is related to T7 RNA polymerase, and the commonly used E. coli BL21 strain contains T7 RNA polymerase gene, we eventually chose to use E. coli DH5α for the experiment. After transforming pAND into DH5α, we selected a single colony and cultured it overnight. Then, we inoculated the overnight saturated bacterial solution into fresh LB (Amp) at a ratio of 1:100. After culturing for 3.5 hours, it entered the logarithmic phase and was divided into four groups. After that, induction was carried out, and four different induction conditions were used in the four groups (Fig. 3). Group 1 did not undergo any operations. The second group was only treated with a one-hour 42°C water bath, the third group was only treated with arabinose (final concentration of 10mM) while the fourth group undergo both induction. After the induction, four cultures were placed on a 37°C shaker and cultured at 200rpm for 12 hours. Then, 1 mL of the sample was taken from each group, centrifuged, washed and resuspended with PBS, and the OD600 and mCherry fluorescence signal were measured.
The results were listed in Fig 4. F/OD was used to quantify the expression of mCherry.
The results shows that when two inputs are present, the output show a significantly increase by a fold of 7.66 (compared to negative control group). Meanwhile, dual-input group’s expression is approximately 2.26 and 5.81 times higher than heat-only group and arabinose-only group respectively. So, the results preliminarily verified the function of our single-plasmid AND gate system.
Expression of mCherry overtime
After preliminarily validating the logical functions of pAND, we aimed to track the changes in pAND expression over time. We conducted the following experiments, using DH5alpha as the experimental strain. Following standard procedures, we obtained an overnight saturated culture and then inoculated it into fresh LB at a 1:100 dilution. We cultured it for 3.5 hours until it reached the logarithmic phase. Subsequently, we performed an induction operation, applying a 1-hour 42-degree water bath heat shock and a final concentration of 10 mM arabinose to the experimental group. We then cultured the samples at 37 degrees and 200 rpm, and measured the fluorescence signals and OD600 at 5h, 12h, 16h, and 24h. The F/OD of each sample was calculated, and the data were plotted as the fold change of the experimental group relative to the control group (Fig. 5).
From the trendline shown in Fig. 5, we can see that the expression initially increased and then decreased. After 24 hours of culturing, the expression fold was still larger than 1 but was much lower than the earlier results. This might indicate that the AND gate does not maintain an open status after receiving signals for one time.
It was a pity that we did not conduct further experiments on this finding, but this experiment shows the potential that the AND gate can not only implement the Open function but also implement the Close function. In theory, mRNA and tRNA, which are the intermediates in this AND gate, have a high turnover rate that could help with the Close function.
The Close function further proves this AND module to be an outstanding candidate for drug secretion because it can be controlled.
Periodic heat-shock test
After confirming the basic function, we became curious about whether periodic heat shocks could affect the strength of the output signal. In theory, only during heat-shock treatment can T7ptag be transcribed, so every heat-shock treatment should increase the amount of T7ptag mRNA. However, how the increased amount of mRNA will affect the overall output of the AND gate was unclear. To investigate this, we conducted another experiment with different inputs. We performed periodic heat shock experiments with either a 4-hour or 9-hour interval. The control group underwent only the first round of heat shock treatment. Prior to each subsequent round of heat shock, fresh LB broth (without interfering with the arabinose concentration) was added. Every heat-shock treatment involved a 1-hour 42°C water bath. Following the heat shock, the cultures were incubated continuously at 37°C and 200 rpm. 1 mL of each group was extracted after 24 hours, counted from the beginning of the first round of heat shock, centrifuged, washed, and resuspended with PBS, and the OD600 and mCherry fluorescence signal were measured. F/OD was measured and graphed (Fig. 6).
The results show that periodic heat shock can indeed increase the expression of the output gene. Meanwhile, a 9-hour interval periodic heat shock worked better than a 4-hour interval periodic heat shock. This may indicate that shorter intervals are not always better, although periodic heat shock did improve expression. It is possible that heat shock interferes with the growth of engineered bacteria, which affects the overall expression.
Different induction conditions
To further understand how inputs affect the output signal, we performed more experiments. We set up additional experimental groups and applied various induction conditions (Fig. 7). We varied the duration of the heat-shock treatment and the final concentration of arabinose. The other procedures were the same as in the previous experiments. Twelve hours after induction, samples were extracted and tested. In this experiment, F/OD was also used as a measurement, but it was normalized to the control group (0 mM arabinose, no heat-shock treatment).
Here's the revised paragraph with corrections and explanations for each change:
As we can see from Fig. 7, the AND gate module behaves differently when facing different amounts of output. Overall, when receiving two outputs, the expression of the output is higher.
When the heat-shock duration is controlled, the expression of mCherry increases as the arabinose concentration elevates. When the arabinose concentration is controlled, the expression of mCherry in the 30-minute heat-shock group is higher than in the 45-minute or 60-minute group. This contradicts our expectation, but it could be related to the overall expression rate of engineered bacteria. In our validation of the PL-PR promoter ( BBa_K4634017), the expression increases 86-fold after a 42°C heat shock compared to 37°C. Thus, T7ptag is heavily transcribed under 42°C heat shock. The large accumulation of mRNA and defective protein (caused by premature translation termination) may trigger the engineered bacteria's regulation circuit and reduce gene expression.
Here's the revised paragraph with corrections and explanations for each change:
Overall, the group subjected to a 30-minute heat shock and 30mM arabinose has the highest expression. Another result to be noticed is that the 30-minute heat shock group works better than the 45-minute or 60-minute group, which might indicate that the most effective heat-shock treatment could be of short duration.
The variation in expression amounts gives the AND gate the potential to be regulated by changing the amount of inputs and provides us with insights to design our heat-shock treatment.
References
[1] Anderson JC, Voigt CA, Arkin AP. Environmental signal integration by a modular AND gate. Mol Syst Biol. 2007;3:133. doi: 10.1038/msb4100173. Epub 2007 Aug 14. PMID: 17700541; PMCID: PMC1964800.| None |