Cycle 1

Cycle 1

Cycle1: LdtR Expression Strain Construction

In order to research LdtR and the functions of our genetic circuit, E. coli instead of L. crescens was used as the model organism because of its short reproduction time, convenient transformation, and known expression system. Therefore, in the first engineering cycle, we attempted to introduce the transcriptional factor of interest, LdtR, into E. coli BL21 (DE3) to construct the Lcr-mimicing strain for further research.

Cycle 1

Design

To yield a stable and massive expression, E. coli strain BL21 (DE3) was selected as the chassis organism, while vector pRSFDuet-1 was selected as the expression vector, the T7 promoter of which is expected to be activated vigorously by T7 RNApol of the strain. Retrieved from open Lcr genome dataset, LdtR gene sequence was codon-optimized for success expression in E. coli. Also, HA tag was fused to its N-terminal for easier detection.

Cycle 1

Build

The optimized LdtR gene was inserted between NcoI and BamHI sites of pRSFDuet-1 vector to become pRSF-LdtR, which was confirmed by DNA sequencing data. Kanamycin selection yielded colonies with success transformation of pRSF-LdtR.

Cycle 1

Test

In order to investigate whether LdtR introduction can stably produce wanted LdtR protein, western blot assay was employed to detect the LdtR-HA fusion protein with the target of HA tag. After induction at 25°C with IPTG, strong signal was detected with HA-tag primary antibodies.

Cycle 1

Learn

The constant expression in transformed strains indicated constitutive, stable, and massive expression of LdtR in our strain BL21-LdtR, which can be used for further studies.

Cycle 2

Cycle 2

Cycle2: Promoter Sensor Construction

We attempted to obtain an accurate and efficient element that specifically react to LdtR so that the engineered phage only release its effects in CLas bacteria. Previous studies showed that promoters of LdtP and ZnuA2 have regulatory roles under LdtR. Therefore, we obtained the two promoters as sensor candidates. After analysis of published whole transcriptome sequencing dataset, we identified a gene of unknown functions, the ID of which was B488_05770, that was down-regulated vigorously when LdtR was inhibited. This suggests that the promoter of B488_05770 might be exclusively activated by LdtR. Therefore, P_B488_05770 was also selected as the sensor candidate of the overall genetic circuit.
The genome of CLasMV1 is a double stranded circular DNA containing 8 genes. Despite functions of most genes are unknown, the gene 1 was identified to code for its capsid protein, which is essential for phage assembly, recognition, and invasion. Due to the limit of whole phage genome materials, we attempted to selected the capsid protein gene as a represent and cloned it into a phage-derived plasmid pBluescript II to mimic the engineered phage DNA. By placing our genetic circuit under the control of candidate sensors, we could optimize the promoter choice for an accurate and effective sensor.

Cycle 2

Design

After obtained from open datasets, the CLasMV1 capsid protein sequence was codon-optimized for success expression in E. coli. Capsid protein gene was fused with FLAG tag for easier detection, which was placed after the candidate promoters (P_LdtP, P_ZnuA2, P_B488_05770). The whole cassette was placed into vector p_Bluescript II KS(+) to yield a engineered phage model, which would be further transformed into BL21-LdtR for detection.

Cycle 2

Build

The sensor-capsid protein expression cassettes were cloned between PciI and HindIII sites of pBluescript II KS(+) vector to become p_BA, pLP, and p_ZA, which were confirmed by DNA sequencing data. Ampicillin and kanamycin double selection yielded colonies with success transformation of the phagemids into BL21-LdtR.

Cycle 2

Test

The sensor-capsid protein expression cassettes were cloned between PciI and HindIII sites of pBluescript II KS(+) vector to become p_BA, pLP, and p_ZA, which were confirmed by DNA sequencing data. Ampicillin and kanamycin double selection yielded colonies with success transformation of the phagemids into BL21-LdtR.

Cycle 2

Learn

The sensor-capsid protein expression cassettes were cloned between PciI and HindIII sites of pBluescript II KS(+) vector to become p_BA, pLP, and p_ZA, which were confirmed by DNA sequencing data. Ampicillin and kanamycin double selection yielded colonies with success transformation of the phagemids into BL21-LdtR.

Cycle 3

Cycle 3

Cycle3: Signal Amplifier Construction

After picking the potential sensors for LdtR recognition of CLas, we attempted to amplify the signal for more efficient production of downstream genes. RinA was identified as an strong activator of its target promoter and has been applied in genetic engineering as an amplifier in previous works, which was selected as the potential amplifier for construction of the sensor-amplifier-downstream gene expression cassette.

Cycle 3

Design

Based on the phagemid in the previous cycle, we cloned the RinA gene under the control of selected sensors (P_LdtP, P_B488_05770). To quantify the amplification efficiency, we placed AcGFP as reporter after the target promoter region of RinA.

Cycle 3

Build

ThThe sensor-amplifier-GFP expression cassettes were cloned between PciI and BamHI sites of pBluescript II KS(+) vector to become pBAG and pPAG, which were confirmed by DNA sequencing data. Expression cassette of sensor-GFP was also constructed for control. Ampicillin and kanamycin double selection yielded colonies with success transformation of the phagemids into BL21-LdtR.

Cycle 3

Test

In order to quantify the amplification efficiency, we measured fluorescence intensity for GFP expression index with or without amplifier. After 24 hours induction, the expression under the sensor P_B488_05770 was dramatically elevated by RinA amplifier, with an almost 100-fold increase in fluorescence intensity. However, only a little enhancement was observed by RinA amplification system under the sensor P_LdtP.

Cycle 3

Learn

In order to quantify the amplification efficiency, we measured fluorescence intensity for GFP expression index with or without amplifier. After 24 hours induction, the expression under the sensor P_B488_05770 was dramatically elevated by RinA amplifier, with an almost 100-fold increase in fluorescence intensity. However, only a little enhancement was observed by RinA amplification system under the sensor P_LdtP.

Cycle 4

Cycle 4

Cycle4: MaSAMP Expression Strain Construction

Research conducted by Huang et. al in 2021 examined the effectiveness of MaSAMP on different types of bacteria, showing the most toxicity on α-proteobacteria, especially L. crescens that they used as model organism in place of Candidatus Liberibacter asiaticus (CLas). E. coli, which is γ-proteobacteria, were tested to show less sensitivity to MaSAMP . Therefore, we decided to use E. coli BL21 (DE3) to express MaSAMP.

Cycle 4

Design

Research conducted by Huang et. al in 2021 examined the effectiveness of MaSAMP on different types of bacteria, showing the most toxicity on α-proteobacteria, especially L. crescens that they used as model organism in place of Candidatus Liberibacter asiaticus (CLas). E. coli, which is γ-proteobacteria, were tested to show less sensitivity to MaSAMP . Therefore, we decided to use E. coli BL21 (DE3) to express MaSAMP.

Cycle 4

Build

We constructed our recombinant plasmid by inserting our designed sequence into pET-30a (+) at the Ndel & Xhol restriction enzymes site. It was then transformed into E. coli BL21 (DE3), amplified and induced by IPTG for massive expression.

Cycle 4

Test

After collecting our constructed strain, we both observed it directly under fluorescent microscope and did western blot after ultrasonication to confirm the expression our fusion protein.

Cycle 4

Learn

MaSAMP along with fusion protein can be expressed in E. coli BL21 (DE3) in a high level. The SUMO protein was confirmed to be an effective tag to hide MaSAMP toxicity.

Cycle 5

Cycle 5

Cycle5: Adjust 6xHis-tag position in recombinant plasmid for better MaSAMP biosynthesis

Adjust 6xHis-tag position in recombinant plasmid for better MaSAMP biosynthesis 6xHis-tag is an important element often added at the N- or C-terminus of the protein for the affinity purification for recombinant protein. In engineering cycle 1, which we used the recombinant plasmid of AMP group, we added the 6xHis-tag at the C-terminus of our fusion protein, right after MaSAMP. However, this 6xHis-tag could not be removed from MaSAMP after SUMO protease cleavage, and it might have the potential to influence the sterilizing effect of MaSAMP. Therefore, designed a new plasmid (named FAMP) by adjusting the 6xHis-tag position. We would then compare the purification process of AMP and FAMP group and also the sterilizing effect of two peptides (with and without 6xHis-tag) released.

Cycle 5

Design

6xHis-tag was moved from the C-terminus of MaSAMP to the position between GFP and SUMO tag since it would be removed along with SUMO tag after SUMO protease cleavage.

Cycle 5

Build

The modified sequence based on the design was inserted into pET-30a (+) at the Ndel & Xhol restriction enzymes site, and then transformed into E. coli BL21 (DE3), amplified and induced by IPTG for massive expression.

Cycle 5

Test

After conducting fusion protein extraction and purification, we did SUMO protease cleavage to release our MaSAMP in both groups. However, the concentration of fusion protein is very low in the supernatant of lysate after centrifuging, making the SUMO protease cleavage hard.

Cycle 5

Learn

After conducting fusion protein extraction and purification, we did SUMO protease cleavage to release our MaSAMP in both groups. However, the concentration of fusion protein is very low in the supernatant of lysate after centrifuging, making the SUMO protease cleavage hard.

Cycle 6

Cycle 6

Cycle6: Improve MaSAMP for better sterilizing effect

The net positive charge and the amphiphilic (hydrophilic and hydrophobic) α-helical are two typical characteristics of antimicrobial peptides (AMP). This is a general conclusion of AMP, but the net positive charge does not fit our AMP. In addition, AMP normally has only one amphiphilic (hydrophilic and hydrophobic) α-helical. The common method of increasing sterilizing effect of AMP is to use lysine to substitute every amino acid. Considering our MaSAMP consists of 67 amino acids including two alpha helixes, it may not be suitable for us to screen every mutant. By referring to the improvement of stapled amino acids, we found the tool for calculating lyticity and the conclusion that substituting amino acids with lysine at the hydrophilic face will increase the sterilizing effect. We came up with the idea of developing a tool that can help us automatically improve amino acid sequence and calculating biophysical parameters, such as total hydrophobicity, net charge and 3D hydrophobic moment vector.

Cycle 6

Design

The principle of improving is to substitute amino acids in the range at the hydrophilic face with lysine. To avoid excessive calculation, we set the substituting range from the 5 amino acids before the alpha helix to the 5 amino acids after the alpha helix. Biophysical parameters, such as total hydrophobicity, net charge and 3D hydrophobic moment vector, are calculated for reference.

cycle 6

Build

Briefly, we used alphafold 2 to predict the protein structure, saving the pdb file and output the position of alpha helix. For the downstream, we used python packages or designed tools to calculate biophysical parameters. Connections between each part were made.

Cycle 6

Test

6 mutants of the main function part of MaSAMP were chemically synthesized to test the potentially increased effect. We will use fluorescent staining to visualize the results.

Cycle 6

Learn

The calculated biophysical parameters can only be used for reference. To fully prove the accuracy of our one-step assembled toolbox, we will further use it to create a mutant library to adapt the principle of our improvement and then narrow the substituting range.

Cycle 7

Cycle 7

Cycle7: Primer construction for gene amplification from D. citri DNA sample

To obtain gene silencing materials, it is necessary to clone native genes from D. citri, which requires construction of corresponding primers. In this cycle, the common genome of D. citri in NCBI was used as reference.

Cycle 7

Design

To design the primers, we searched for our target gene sequences (DCR, EFE4, CP450) in the National Center for Biotechnology Information (NCBI) system. We designed the primers based on the mRNA sequence of the genes without the restriction enzyme sites to confirm the amplification of the gene. Then, we designed the primers with restriction enzyme sites to amplify both the antisense and sense direction of target genes.

Cycle 7

Build

For the sense fragment, we utilized two different restriction enzyme sites for each of the forward and reverse primers. The same strategy is utilized for building the antisense fragment. As a whole in the L4440 vector system, there will be a linker in between the sense and antisense fragments, so that when it is transcribed, it will form a hairpin RNA.

Cycle 7

Test

We tested the primers by using polymerase chain reaction (PCR) using the extracted D. citri DNA as the DNA template. Then, we ran gel electrophoresis to confirm the amplification. The gel result shows that the three target genes were successfully amplified. We then ran the PCR using the primers with the restriction enzyme sites. The result showed successful amplification of the CP450 gene with restriction enzyme sites for sense and antisense directions. However, the EFE4 gene was not amplified. We moved on to cloning with the CP450 gene.

Cycle 7

Learn

From the gel result, we learned that the genes matched the expected fragment length (405 bp for EFE4 and 412 bp for CP450). DCR gene had introns between the forward and reverse primers, resulting in a longer fragment (800 bp) than expected (275 bp). Therefore, we extracted RNA from D. citri, converted it into a cDNA, and ran the PCR with the cDNA template, which identified the correct amplification of the DCR gene in both sense and antisense directions. We tried to troubleshoot for amplification of EFE4 with primers with restriction enzyme sites by trying different annealing temperatures and using, but it was not successfully amplified, which may be due to the long length of the primer.

Cycle 8

Cycle 8

Cycle8: Cloning of CP450 gene onto the L4440 plasmid

In our next cycle, we tried to clone one of our target genes onto the L4440 plasmid, which was confirmed of a successful amplification by engineering cycle 1

Cycle 8

Design

To utilize the HT115-L4440 RNAi production method, we tried to clone the amplified fragment of sense and antisense strand CP450 onto the L4440 plasmid vector.

Cycle 8

Build

We first tried to digest the CP450 antisense PCR fragment and the L4440 plasmid with antisense restriction enzymes, XbaI and SacI. To confirm successful digestion, we planned to confirm with a gel by running the digested PCR product and plasmid. Then we tried to ligate the digested PCR product and plasmid together. We transformed the ligated product into the E.coli DH5a strain to screen for the vector with successful ligation and an empty vector. After miniprep, we confirmed successfully ligated vector with PCR using the ligated plasmid vector as the template and using CP450 gene primers.

Cycle 8

Test

According to the gel result, it showed that the digested vector bands had a larger band size than the undigested vector. For the PCR digestion, we did not see a clear band on the gel. After ligation, digestion, miniprep, and PCR, the gel result showed an amplification of the CP450 gene for one of the plasmids we tested, indicating successful ligation of the CP450 antisense gene onto the L4440 vector.

Cycle 8

Learn

The first gel result shows that both restriction enzymes work well on our vector, since we can see that the band of the uncut vector shows a smaller band size than the digested vector. This indicates that the digested vector is a linear form of the plasmid rather than a circular, undigested one. The vector cloned with CP450 antisense could be used for further cloning of CP450 sense fragment to make a complete hpRNA.

Cycle 9

Cycle 9

Cycle9: Increasing the production of hpRNA using additional T7 promoters

In this engineering cycle, we tried to test if having an additional T7 promoter in our vector L4440 would increase the hpRNA production, using three different target genes of D. citri (MP20, CP64, and DM2).

Cycle 9

Design

In the vector L4440 which already has a T7 promoter, we added an additional T7 promoter sequence to the target gene sequence. The T7 and three target gene sequences were successfully cloned into the plasmid via a commercial biotech company. We then planned to test the efficiency of the three genes without additional T7 promoters and those with additional T7 promoters utilizing the HT115(DE3) E. coli bacterial strain and IPTG induction.

Cycle 9

Build

We transformed the cloned plasmids into HT115(DE3) E. coli bacteria, and successful transformation was screened with tetracycline and ampicillin antibiotics. Then we cultured the colonies and added IPTG to induce hpRNA transcription. We utilized CTAB miniprep to extract hpRNA and ran the final product on the gel with the treatment with RNase A to leave only the hpRNA produced.

Cycle 9

Test

We tested the final product of RNAi production on the gel. The gel result shows successful induction of hpRNA for samples that were treated with IPTG and RNase A

Cycle 9

Learn

We learned from the gel that it is difficult to observe if the system with additional T7 promoters produced more hpRNA. Therefore, to meet our ultimate goal of testing the efficiency of hpRNA production under an additional T7 promoter system, we need to measure the RNA quantity using the Nanodrop method.