Project-Engineering

Design

We aim to develop light-induced E. coli that can produce L-dopa. Therefore, we have to build two systems: the fermentation system and the regulation system.

Fermentation system

Levodopa, an amino acid prevalent in neurons for being the precursor of dopamine, is not rich in bacteria because it doesn’t construct proteins. However, its precursor, tyrosine, is abundant in bacteria for its crucial functions. Therefore, we have paid a great amount of efforts to work on this crucial problem.

In this reaction, by consuming oxygen and FADH2, levodopa, a monophenol, is oxidized into diphenol under the catalyst of enzymes. Also, a flavin reductase must exist in the reaction, since this reaction consumes a large amount of FADH2 to prevent diphenol from being oxidized into quinone and later useless melanin【1】. HpaC is a flavin reductase which fit the demand. Since HpaB locates next to HpaC【2】, we replicate the DNA fragment in a concatenation way, which includes sequences of HpaB and HpaC and their RBSs. Then, to boost the yield, the fragment is assembled into a plasmid with a higher copy number.

Figure 2: Mechanism of HpaBC catalyzes Tyrosine to Levodopa

Regulation System

T7 promoter is highly specific because only T7RP (T7 RNA Polymerase) can combine with it, and the transcription will then be induced. A sequence which expresses HpaB and HpaC is designed to follow the T7 promoter. Thus, we can regulate the yielding simply by regulating the activation of T7RP. Since we aim to build a light-induced fermentation system, we use some light-induced allostery proteins (Vvd and Mag)【3】 to regulate the activation of T7RP. These light-induced allostery proteins can respond to blue light. To express the enzyme, sequences of split T7 RNA polymerase are assembled into a plasmid which has a low copy number. Sequences of photoswitches (light-sensitive proteins) are assembled between the split-sequence with a link with T7 RNA polymerase to achieve the light regulation. Therefore, split T7RPs are expressed and float in the cytoplasm. When the blue light is on, the photoswitches will transform their structures and aggregate with each other. After their match, T7RPs pair into pairs and combine with the T7 promoter. At last, the sequence after the T7 promoter is transcribed and HpaB and HpaC will be translated continuously. This starts the fermentation system.

The Combination of systems

The fermentation system and the regulation system are separated on two different plasmids. Since the regulation system expresses T7RP, we give the system a smaller copy number. On the other hand, the plasmid of the fermentation system (pHpaBC) has a larger one. This is to build a balance in producing levodopa. We transformed both plasmids into E. coli and get a combined system. When the blue light on, T7RP complexes form by aggregation of activated photoswitches. This triggers transcriptions. After the HpaB and Hpac are expressed, the L-dopa yielding begins. Therefore, the regulation system achieves switching on in the presence of blue light, and the fermentation system successfully converts Tyrosine to L-dopa. This enables us to precisely controls the levodopa dose.

[1]: Xun, L. and Sandvik, E., 2000. Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase. Applied and Environmental Microbiology, 66(2), pp.481-486. [2]: Jeong, H., Kim, H. and Lee, S., 2015. Complete Genome Sequence of Escherichia coli Strain BL21. Genome Announcements, 3(2).
[3]: Kawano, F., Suzuki, H., Furuya, A. and Sato, M., 2015. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nature Communications, 6(1).

The enzymes that produce levodopa are independent. HpaB and HpaC, sometimes are called HpaBC. Though amino acids sequences of HpaBC are the same, two different DNA sequences, wildtype (WT) and SMS are obtained. WT is derived from the BL21 genome by PCR, and SMS is artificially synthesized with code optimization. The two sequences are separately assembled to a vector pET28b to replace the original GFP by Gibson assembly. Therefore, we derive two similar plasmids that can both be regulated in transcription by light but might have different effects in translation.

Our light-induced system regulates the fermentation system by regulating T7RP (T7 RNA Polymerase). Therefore, we prepared the fragments of P70 vector, photoswitches, and T7 RNA Polymerase by PCR. Some RBS, promoter, and BsaI sites are added by PCR. For photoswitches, we selected three: pMag-nMag, pMag Fast 2-nMag High 1, Vvd. Since we need T7RP to work when and only when light is applied, we need to divide the sequence of T7RP into two sites. We selected 179[1] and 564[2] as the sites. To achieve the regulation system, we insert photoswitches between two divided sequences of T7RP. All fragments are assembled by Goldengate assembly.

Culture in different conditions

To test the efficiency of the light regulation system, we applied the photoswitches efficiency test. We create two groups in the test, one with blue light on and another in dark. The group with blue light (BL group), is applied no modification. In another group, which is light-free, is cover by tin foiled for shadowing. An additional group, the experiment group, is exposed to light for the first 5 hours and a later time with light-off.

Detailed processes:
1. Overnight bacteria culture is inoculated into fresh LB medium with a proportion of 1:200.
2. Positive and experiment groups are exposed in blue light and the negative group remained completely covered with tin.
3. Samples are overnight cultivated in 37℃, 220rpm. Samples are taken out after 24 hours’ cultivation.
4. All samples are taken to fluorescence measurement, which we will talk about later in the Measurement(link to the section) section.
5. the whole experiment takes about 24 hours.

To examine the efficiency of HpaBC-WT and HpaBC-SMS and to confirm the practicability of our fermentation system, we first prepare DH10b[3], a strain that is integrated with T7RNAP constitutive expression unit, as component cell. Then we transform HpaBC-WT and HpaBC-SMS to component cells, respectively. The monoclonals are later overnight cultivated, and is then inoculated into fresh LB medium with a proportion of 1:200. The samples are then cultivated for 48h, in 37℃, 220rpm. Samples are taken out, after the 48-hours-long cultivation, to measure for its L-dopa concentration. The detailed description of measurement is illustrated below(link to determine the standard curve of levodopa).

After our test on photoswitches’ efficiency, we still need further data on our light-induced fermentation system. Therefore, we apply a similar test on the bacteria (DH10b), which contained both plasmids described above. Unlike the last test, the experiment group is deleted. All samples are shadowed or exposed until the end. From this test, we expect to get more direct data about our system.

Detailed processes:
1. Inoculate overnight bacteria culture into fresh LB medium with a proportion of 1:200.
2. Light group is exposed in blue light and the Dark group remains covered.
3. samples are taken only on several points: 8h, 16h, 24h, 28h, 32h, 44h.
4. after the experiment end, all samples are taken to levodopa measurement, which we’ll talk about later in the Measurement section.
5. the whole experiment takes about 46 hours.

[1]: Shis, D. and Bennett, M., 2013. Library of synthetic transcriptional AND gates built with split T7 RNA polymerase

mutants. Proceedings of the National Academy of Sciences, 110(13), pp.5028-5033.

[2]: Han, T., Chen, Q. and Liu, H., 2016. Engineered Photoactivatable Genetic Switches Based on the

Bacterium Phage T7 RNA Polymerase. ACS Synthetic Biology, 6(2), pp.357-366.

[3]: Zong, Y., Zhang, H., Lyu, C., Ji, X., Hou, J., Guo, X., Ouyang, Q. and Lou, C., 2017. Insulated transcriptional

elements enable precise design of genetic circuits. Nature Communications, 8(1).

Team:SMS Shenzhen/Engineering

Project-Engineering

Design

Fermentation system