Our experiments are divided into seven parts, mainly based on molecular biology, but also involve knowledge from other disciplines. We strive to achieve initial validation of the project design.

Module 1 Basic fermentation

Since three plasmids were obtained from the laboratory of teacher Qipeng Yuan of Beijing University of Chemical Technology, we planned to transform the three plasmids into DH5α receptor strains in the first module of basic fermentation experiment. The three groups are one plasmid pSA-ARO10-adh6 and two plasmids pSA-ARO10-adh6, pET-UGT85A1 and three plasmids pSA-ARO10-adh6, pET-UGT85A1, pCS-pgm-galu. After transformation, the electrophoretic results of colony PCR were shown in Figure 1.1.

Figure1.1 Three plasmid colony PCR results (pSA-ARO1010-adh6 validation on left 1-4, pET-UGT85A1 validation on left 5-8, and pCS-pgm-galu validation on left 9-10)

After colony PCR verification was correct, we carried out fermentation experiment. Single colony was cultured overnight before test tube, and OD value was measured to ensure that OD value was the same as 0.05 when inoculated into shaker, and OD value was cultured to 0.3-0.4 in shaker, and inducer was added. Samples were taken at 12h, 24h and 36h respectively. Standard sample solutions with different concentrations were configured. We used water and M9 medium as solvents, respectively, and the concentration gradients were set to 25,50,100,200 mg/L.

The specific data of the liquid chromatography of the standard sample solution are shown in Table 1.1, the standard curve is shown in Figure 1a, and HPLC analysis of salidroside standard is shown in Figure 1b and Figure 1c . The detailed data of fermentation results of strains after transformation of different plasmids are shown in Table1.1, and HPLC analysis is shown in Figure 1d and Figure 1e .

Table 1.1 Detailed data of HPLC analysis (Contains standard products and samples)

Fig.1 (a) It is a standard curve drawn according to HPLC data obtained from salidroside standard with M9 as the solvent in Table1.1, which is used to calculate the concentration of salidroside in subsequent samples. (b) and (c) were obtained by HPLC analysis of salidroside standard in water and M9 medium, respectively. (d) HPLC analysis of the fermentation results of the group 2 (see Table 1.1 for details of the group). The sampling time represented from left to right was 24h and 36h.(e) HPLC analysis of the fermentation results of the group 3 (see Table 1.1 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.

According to the standard curve, there was no salidroside peak in the test results of one plasmid group, the highest concentration of salidroside in the test results of two plasmid groups was 7.2mg/L, and the highest concentration of salidroside in the test results of three plasmid groups was 12.3mg/L. Since the function of pSA-ARO10-adh6 is to convert 4-hydroxypheny lpyruvate to tyrosol, it is only transferred to one plasmid group without salidroside production. pET-UGT85A1 encodes glycosyltransferase, which converts tyrosol into salidroside, so salidroside can be detected by switching into two plasmid groups, as shown in Figure 1d. pCS-pgm-galu encodes glucose-mutase and UDP-glucose-transferase, which can regulate metabolic flux to increase yield, and the comparison of the second and third groups shows its efficacy.

Module 2 Copy Number Optimization Module

2.1 Original Vector Swap

Since the original vectors have different copy numbers, we tried to search for the optimal copy number combination for rhodioloside production by changing the vectors on which the genes are installed.

We use the original plasmids pSA-ARO10-adh6, pET-UGT85A1 and pCS-pgm-galu to obtain our backbones and genes. Then the backbones and genes are purified through gel electrophoresis and ready for enzyme processing. Small volumes of product are sampled for verification,as shown in Figure 2a.

The backbones and genes are processed for sticky ends and then linked by T4 DNA ligase. From this we built six new recombinant plasmids: pSA-UGT85A1, pSA-pgm-galu, pET-ARO10-adh6, pET-pgm-galu, pCS-ARO10-adh6, pCS-UGT85A1, and transferred them into DH5α.

Colonies of recombinant plasmid strains are selected and went through verification PCR. Vector Swap section have been performed multiple times for its relatively low possibility of success. Results indicate that most recombinant plasmid strains have been successfully constructed,as shown in Figure 2b and Figure 2c.

Fig.2 (a)Verification for backbones and genes. Right side of the marker lane is the products gone through enzyme processing to form sticky ends (see 1.2 below) while the left side unprocessed for comparison. On both sides from left to right: Lane 1: pSA Backbone; Lane 2: ARO10-adh6; Lane 3: pET Backbone; Lane 4: UGT85A1; Lane 5: pCS Backbone; Lane 6: pgm-galU.(b)(c) Verification for recombinant plasmid strains. Strains with positive results are supposed to have a band around 1200. (b) Lane 1 to 4: pSA-UGT85A1; Lane 5 to 8: pSA-pgm-galU; Lane 9 to 12: pCS-ARO10-adh6; Lane 13 to 16: pCS-UGT85A1; Lane 17: Marker. (c) Lane 1 to 4: pSA-UGT85A1; Lane 5 to 8: pSA-pgm-galU; Lane 9 to 12: pET-ARO10-adh6; Lane 13 to 16: pET-pgm-galU; Lane 17 to 20: pCS-ARO10-adh6; Lane 21 to 24: pCS-UGT85A1.

2.2 Dual Expression Vector Installation

The original plasmids all have relatively low copy numbers, which is supposed to affect our rhodioloside production. Therefore, we researched on vectors with high copy numbers and high expression efficiency.

The new vectors we use are dual expression vectors pACYCDuet, pCDFDuet, and pETDuet, given by Prof. Yu Hongwei’s research group. We use reverse PCR to obtain their backbones. Genes are obtained through PCR using primers designed compatible for Gibson Assembly, with original plasmids as the template.

Small volumes of PCR products are used for verification,as shown in Figure 3a. The rest is processed by DpnI to eliminate the template and then purified.

Concentrations of purified backbones and genes are measured to prepare for the reaction. Recombinant plasmids were constructed through Gibson Assembly.

Colonies of recombinant plasmid strains are selected and went through verification PCR,as shown in Figure 3b.

Only one single gene has been installed on one of the expression sites. By installing another gene based on the plasmid repeating the protocol, we could construct the full dual expression vector. After discussion, we finally chose pACYCDuet vector and constructed the dual expression plasmid pACYCDuet-ARO10-adh6-UGT85A1,as shown in Figure 3c and Figure 3d.

The recombinant plasmids, either only one or both genes installed, went through sequencing using the verification primers. The sequence of recombinant plasmids has been confirmed, then transformed into chassis to construct engineered fermentation strains,as shown in Figure 3e.

Fig.3 (a)Verification of PCR Products ready for Gibson Assembly®. Lanes 1 & 2: ARO10-adh6; Lanes 3 & 4: pCDFDuet Backbone; Lane 5: pETDuet Backbone; Lane 6: UGT85A1; Lanes 7 & 8: pACYCDuet Backbone. (b)Verification for recombinant plasmid strains. Lane 1 to 4: pACYCDuet-UGT85A1; Lane 4 to 8: pCDFDuet-UGT85A1; Lane 9 to 12: pETDuet-UGT85A1. (c)Engineered Dual Expression Plasmid pACYCDuet-ARO10-adh6-UGT85A1. (d)Characterization for plasmid pACYCDuet-ARO10-adh6-UGT85A1. Lane 1-8: ARO10-adh6 verification; Lane 9-16: UGT85A1 verification. (e)Sequencing result for pACYCDuet-ARO10-adh6-UGT85A1 indicating the successful assembly of the recombinant plasmid.

We constructed the following recombinant strains that has changed the copy numbers and researched on the fermentation results: 4.pACYCDuet-UGT85A1 + pSA-ARO10-adh6 5.pACYCDuet-UGT85A1-ARO10-adh6

After the verification has succeeded,as shown in Figure 4a and Figure 4b , the strains are inoculated for fermentation. Fermentation samples are measured through HPLC and finally calculated into concentrations from standard curve respectively,as shown in Figure 4c and Figure 4d .

By comparing type 4 with original strain pSA-ARO10-adh6 + pET-UGT85A1, as shown in Table 2.1,we could conclude that only by increasing one copy number could lower the yield instead, which may result from the higher metabolic stress by pACYCDuet plasmid, or the side reactions caused by excessive UGT85A1 enzymes.

By comparing type 5 with type 4, as shown in Table 2.1,we could conclude that using high-copy double expression could increase the rhodioloside yield but on a very limited level. Possible reasons are that the strains are given high expression and metabolic pressure from pACYCDuet. With abundant resources used for synthesizing the plasmid and express the enzymes, the cells have less left for growth as well as rhodioloside production. The notably low OD value of type 5 among all fermentation strains could also be a supporting fact for the result.

Among these fermentation strains, there always has a much higher peak on peak time around 18.0,as shown in Figure 4e. And it is observed that the more UGT85A1 expression activity overwhelms that of ARO10-adh6, the higher this peak gets. In the control strain that only expresses UGT85A1, the peak gets the highest. This indicates that side reaction caused by the expression mismatch of UGT85A1 and tyrosol is one of the fatal factors affecting our rhodioloside yield.

In conclusion, we confirmed that the increase of copy number could increase the rhodioloside yield. However, it also brings side effects like affected growth, abundant side reactions, etc., which have limited our rhodioloside yield. We could infer that there could have an optimal copy number match that could eliminate the bottlenecks, producing rhodioloside at a highest yield.

Table1.2 Detailed data of HPLC analysis(Reconstruct the plasmid set)

Fig.4 (a)Verification for fermentation strains. Lane 1 to 8: type 4 for ARO10-adh6; Lane 17 to 24: type 4 for UGT85A1. (b) Lane 1 to 8: type 5 for ARO10-adh6; Lane 9 to 16: type 5 for UGT85A1.(c)HPLC analysis of the fermentation results of the group 4 (see Table 1.2 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.(d)HPLC analysis of the fermentation results of the group 5 (see Table 1.2 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.(e)HPLC analysis of control strain pCS-UGT85A1 on 36h. Notably high peak around time 18.0.

Module 3 Gene knockout module

We used CRISPR-Cas9 for gene knockout, and prepared Donor fragment containing 500bp length at each end of the target gene to achieve seamless knockout through homologous recombination. Two plasmids are involved, namely pTarget (BBa_K4761107) and pCas containing Cas9 gene (BBa_K4761006). The pTarget plasmid can express sgRNA.

We used the website (CHOPCHOP.cbu.uib.no) to design sgRNA targeting pykA, pykF and pheA genes (BBa_K4761020-BBa_K4761028). pTarget plasmid containing target sgRNA (BBa_K4761420-BBa_K4761428) was obtained by rPCR, namely the sgRNA expression box. In order to improve the purity, we treated the rPCR products with the methylation template digestion enzyme DpnI, digested the pTarget plasmid template, and then cleaned and purified the sgRNA expression box with high purity.We successfully constructed pTarget plasmid containing target sgRNA, and the sequencing results were shown in Figure 5a.

We designed polymerase chain reaction primer P1-P4 (BBa_K4761520-BBa_K4761531) for Donor DNA preparation on the upstream and downstream of pheA, pykA and pykF genes of Escherichia coli, respectively. The 500bp DNA fragments of pheA, pykA and pykF genes on the genome were obtained by the first round of polymerase chain reaction, and then fusion PCR was performed to integrate into Donor DNA of pheA, pykA and pykF genes with a length of 1000bp,as shown in Figure 5b.

By means of receptor transformation, pCas plasmid containing Cas9 gene (BBa_K4761006), pTarget plasmid expressing sgRNA, and Donor DNA were successively transformed into E. coli DH5α cells.

We had planned to knock out all three genes in sequence within one E. coli strain. However, due to the impact of CRISPR-Cas9 gene knockout efficiency, our gene knockout experiments for pykA, pykF and pheA failed many times. To this end, we redesigned their sgRNA and, to improve efficiency, ran knock-out experiments on three identical E. coli DH5α strains in parallel. We designed validation primers at each side of pykA, pykF and pheA with a length of 1000bp to perform PCR on the obtained colonies. Compared with the control group, the gel electrophoresis band of some strains changed from 3000bp to 2000bp, indicating that the Donor sequence has achieved perfect replacement, and we finally obtained some ΔpykA, ΔpykF, ΔpheA three E. coli gene mutant strains. See Figure 5c,Figure 5d and Figure 5e.

Removal of plasmids.After obtaining three E. coli strain mutants, ΔpykA, ΔpykF and ΔpheA, the presence of exogenous plasmids pCas and pTarget in E. coli was detrimental to the growth of the strain, so we continued the experiment of removing the plasmids. pCas plasmid carries kanamycin resistance gene and pTarget plasmid carries streptomycin resistance gene, so the target strains of corresponding plasmids are screened. We eventually obtained a mutant strain of the target E. coli gene.

Fig.5 (a)The sequencing results of pTarget plasmid indicating the successful assembly of the sgRNA expression boxes targeting pheA, pykA and pykF genes.(b)Verification of Donor DNA preparation (c)pheA gene knockout validation. Positive results should have a band around 2000bp.(d)pykA gene knockout validation. Positive results should have a band around 2000bp.(e)pykF gene knockout validation. Positive results should have a band around 2000bp.

Due to time, we made only ΔpheA and ΔpykA receptive strains, and transferred the two plasmoids pSA-ARO10-adh6,pET-UGT85A1 into the two receptive strains for fermentation experiment, which was the same as module 1.

The corresponding product concentration was calculated according to the standard curve of Figure 1a. See Table 3.1 for detailed data, HPLC analysis of the fermentation results of the group 6 and group 7,as shown in Figure 6a and Figure 6b.

According to the fermentation results, it can be seen that the strain with pykA gene knockout has an increase effect on salidroside production, while the strain with pheA gene knockout has no significant increase. However, according to literature description, pheA gene knockout should greatly improve salidroside production, because pheA gene knockout can block competitive L-phenylalanine synthesis and improve the metabolic flux of salidroside precursors. We speculated that the growth of pheA gene knockout strain was restricted due to the lack of corresponding nutrition, which showed no significant increase in salidroside production.

In summary, gene knockout divided by the regulation of metabolism is conducive to the increase of salidroside production.

Table3.1 Detailed data of HPLC analysis(Gene knockout)

Fig.6 (a) HPLC analysis of the fermentation results of the group 6 (see Table 3.1 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.(b) HPLC analysis of the fermentation results of the group 7 (see Table 3.1 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.

Module 4 Fusion express

We designed three flexible and three rigid joints (BBa_K4761030-BBa_K4761035) to connect the adh6 and ARO1010 genes. The fusion expression essence is that the stop codon of the first gene is replaced by the linker sequence. We finally chose pET (BBa_K4761101) as the skeleton.

First, we used P1 and P2 series PCR from primers No. 3-1 to 3-13 to extract the adh6 gene fragment with the cleavage site and linker sequence, and P3 and P4 series PCR to extract the corresponding ARO10 gene fragment, as shown in Figure 7a.

The adh6 gene fragment corresponding to linker and ARO10 gene fragment corresponding to linker were fused by PCR using P1 and P4 series primers to obtain the combined gene fragment linked by Linker sequence. Electrophoresis results were shown in Fig 7b and sequencing results in Fig 7c. According to the sequencing results, except for the Linker5 sequence, the other five groups were successfully sequenced.

After the correctness of the fragment was verified, the fusion gene fragment was connected to the pET plasmid skeleton by the method of enzyme digestion and enzyme linkage, and colony PCR and sequencing were performed after transformation. Colony PCR results were shown in Figure 7d and sequencing results in Fig 7e. According to the sequencing results, only Linker3 and Linker4 groups were successful.

The two plasmids pSA-ARO10-Linker3-adh6, pACYCDuet-UGT85A1 and pSA-ARO10-Linker4-adh6, pACYCDuet-UGT85A1 were transferred into BL21(DE3) respectively. Firstly, Colony PCR was performed to verify whether the two plasmids were successfully transferred. The electrophoretic results were shown in Figure 7f.

Fig.7 (a)First PCR in fusion PCR.(b)Electrophoresis results after fusion PCR (Linker1-6 from left to right). (c)Results of Linker sequence sequencing.(d)Results of colony PCR electrophoresis.(e)Sequencing results of reconstructed plasmid.(f)The fermentation strains were verified by electrophoresis,Left 1 to left 4 are Linker3 validations, and left 5 to left 8 are Linker4 validations.

Fermentation experiments were conducted after the verification was correct,as shown in Figure 7f. The fermentation results of Linker3 group and Linker4 group were shown in Fig 8a and Fig 8b, and the detailed fermentation data of the two groups were shown in Table 4.1. According to the standard curve Figure 1a, the highest concentration of salidroside fermentation in Linker3 group was 11.6mg/L, and that in Linker4 group was 5.35mg/L. In conclusion, flexible connector LInker3 can significantly increase salidroside yield compared with rigid connector LInker4. This may be related to the special properties of flexible joints.

Table4.1 Detailed data of HPLC analysis(Fusion express)

Fig.8 (a) HPLC analysis of the fermentation results of the group 8 (see Table 4.1 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.(b) HPLC analysis of the fermentation results of the group 9 (see Table 4.1 for details of the group). The sampling time represented from left to right was 12h,24h and 36h.

Module 5 Gene Integration

We design to integrate ARO10 gene and UGT85A1 gene into E. coli using CRISPR RNA-guided integrases and this system involves three plasmids, pEffector (BBa_K4761052), pDonor (BBa_K4761108) and pCutamp. Plasmid pEffector contains specific sgRNA sequences that determine the location of gene integration. Plasmid pDonor amplifies exogenous gene fragments in the organism. And plasmid pCutamp contains genes such as Cas9. Our aim is to insert ARO10 gene and UGT85A1 gene into pDonor and transform these three plasmids into E. coli, and discard the plasmids after integration is completed,as shown in Figure 9a.

Colony PCR was performed with the corresponding confirmation primers, and electrophoresis results showed that we successfully inserted the genes of UGT85A1 and ARO10 into pDonor to construct plasmids pDonor-UGT85A1 and pDonor-ARO10, respectively.

After that, plasmids pDonor-UGT85A1/pDonor-ARO10, pCutamp and pEffector (B28T10) (Amp, Apr, and Sm resistance, respectively) were transfected into DH5α. Colony PCR was performed with the corresponding confirmation primers, and electrophoresis results(Figure 9b) showed that we successfully transfected three plasmids in DH5α.

After incubation at 30°C for 20h, colony PCR was performed with primers designed at both ends of the insertion site on the DH5α genome, and Figure 9c showed that we successfully inserted UGT85A1 and ARO10 respectively into the genome of E. coli DH5α.

Successful groups were partly stored and partly used for subsequent plasmid discarding; we inoculated the compliant strains into LB liquid medium without resistance for overnight incubation and then inoculated them onto plates with three single resistances for incubation (Apr, Amp, and Sm resistance, respectively). In the first round of discarding, some strains successfully discarded two of the three plasmids,as shown in Figure 9d and Figure 9e..

After that, another round of discarding was repeated by using strains U1-1, A2-1 and A2-2 and we aquired U1-1-3 that discarded all of the three plasmids so it couldn’t grow on the plate with Amp resistance, which meant that we obtained strains with successful gene integration and with discarding all of the transferred plasmids,as shown in Figure 9f.

Fig.9 (a)pDonor-ARO10(left)and pDonor-UGT85A1(right)confirmation electrophoresis results (b)Plasmid transformation confirmation electrophoresis results(left 1-4 of pCutamp、left 5-8 of pDonor-UGT85A1/ARO10 and right 1-4 of B28T10).(c)Gene insertion confirmation electrophoresis results(left 1-6 and right 1-3 of ARO10, middle 1-6 and right 4-6 of UGT85A1) (d)(e)Results of the first round of plasmid discarding,U1-1 and A2-2 discarded B28T10 and pCutamp so they only grew on the plate with single Amp resistance; A2-1 discarded pDonor-ARO10 and pCutamp so it only grew on the plate with Sm resistance.(f)Results of the second round of plasmid discarding.

Module 6 RNA thermometers

We obtained the EGFP gene from Professor Xu Zhinan's research group, but it is located in the genome of the strain. Therefore, we designed primers with primer numbers 5-1 and 5-2 to construct the pET-EGFP plasmid, as shown in Figure 10a.

We designed the RNA temperature elements of of U6-1 (BBa_K4761040), U6-2 (BBa_K4761041), U7 (BBa_K4761042), U8 (BBa_K4761043)and U9 (BBa_K4761044), and simulated the secondary structure figure online (http://rna.tbi.univie.ac.at/) , shown in Figure 10b. MFE structure, the thermodynamic ensemble of RNA structures, and the centroid structure, the positional entropy for each position, as shown in Figure 10c.

Single stranded DNA is synthesized and then complementary to form double stranded DNA through heating and slow cooling. The region between the promoter and start codon in pET-EGFP is replaced through enzyme cleavage. U6, U7, and U8 are successfully constructed, as shown in Figure 10d.

Fig.10 (a)pET-EGFP Plasmid Map.(b)Folding simulation of secondary structure of RNA thermometer elements,1 is U6-1, 2 is U6-2, 3 is U7, 4 is U8,5 is U9.(c)MFE structure, the thermodynamic ensemble of RNA structures, and the centroid structure, the positional entropy for each position,1 is U6-1, 2 is U6-2, 3 is U7, 4 is U8,5 is U9.(d)Sequencing comparison results of U6,U7, and U8 after construction.

After the construction is completed, the strain is placed in a test tube culture and the inducer IPTG is added. Firstly, a standard curve over time is plotted to determine the optimal sampling time. Incubate the control group (non fluorescent) and U0 (strain containing pET-EGFP), U6, U7, and U8 at 37 ° C for 8 hours, 16 hours, and 24 hours, and then take samples to detect OD values and Relative FluorescenceUnits (RFU). Plot the RFU/OD-Time curve, as shown in Figure 11a. After comprehensive consideration, 16 hours is chosen as the optimal sampling and testing time.

Afterwards, the control group (non fluorescent) and U0 (strain containing pET-EGFP), U6, U7, and U8 were cultured at 17 ℃, 22 ℃, 30 ℃, and 37 ℃ for 16 hours, respectively, and then sampled for detection. According to literature, the 17 ℃ and 22 ℃ groups were cultured for 6 hours. Please refer to Figure 11b for details. It can be seen that the fluorescence intensity of the U0 group is higher than that of the control group at different temperatures, but the fluorescence intensity will decrease as the temperature decreases. The fluorescence intensity of U6, U7, and U8 groups was significantly higher than that of 17 ℃ and 22 ℃ at 30 ℃ and 37 ℃.

In order to more intuitively observe the construction effect of RNA thermometer, we chose the pET28a EGFP plasmid (BBa_K4761106) to test the RNA thermometer effect. This plasmid contains the T7 promoter, and the above RNA thermometer elements were integrated into the plasmid using the same method, and transformed into the BL21 (DE3) strain for expression detection. The specific results are shown in Figure 11c.The results showed that U6 had a good temperature response and was expressed at 37 ° C, but not at 30 ° C and below. However, U7,U8 and U9 were expressed at 30 degrees and 37 degrees, and had a lower expression level at 30 degrees. The U0 group without the RNA thermometer element expressed at all four temperatures, but the expression level also decreased with the decrease of temperature.

Fig.11 (a)RFU/OD-Time Curve(b)RFU/OD-Temperature Curve (c)A quick comparison of RNA thermometer constructs by temperature-controlled EGFP expression in E. coli

Module 7 Lipsome vesicle

According to the DLS result,as shown in Figure 12, when hydrated with pure water, we obtained lipsomes with sizes around 1200nm, which indicated that we could successfully make lipsomes using our method. When hydrated with LB medium containing E.coli, we additionally found a peak around 5500nm except the peak of about 1200-1500nm, which indicated that we might obtain lipsomes with E.coli inside. And when we changed the ratio of phosphatidylcholine(PC) and cholesterol(CH), we found that increasing the proportion of CH increased the number of lipsomes(at 1200-1500nm and 5500nm), which might indicated that cholesterol could improve the stability of lipsome.

Fig.12 DLS result of lipsome.blue line, PC: CH = 7: 2, hydrated with pure water; red line, PC: CH = 7: 2, hydrated with LB medium containing E.coli; purple line, PC: CH = 7: 4, hydrated with LB medium containing E.coli; yellow line, PC: CH = 7: 6, hydrated with LB medium containing E.coli; the ratios above are mass ratios.

According to Figure 13a, when hydrated with water and PC: CH=7: 2, we found most of the liposomes at 1μm size, with some additional unilamellar vesicles of 5μm size and multilamellar vesicles of 10-20μm size. This indicated that we successfully obtained lipsomes of suitable sizes.

According to Fig Figure 13b and Figure 13c, we found most of the liposomes of 5μm size(some of them were multilamellar vesicles) and some lipsomes of about 1μm size. Also, there were some lipsomes containing E.coli inside according to Figure 13c. These proved that we obtained lipsomes capable of encapsulating E.coli and cross-corroborated our DLS result.

We could also spot multilamellar vesicles of around 5-10μm size, according to Figure 13d and Figure 13e, when PC:CH=7:4 and PC:CH=7:6. However, compared to PC:CH=7:2, these two figures showed less lipsomes and we failed to find lipsomes that encapsulated E.coli, which might indicated that although cholesterol could improve the stability of lipsomes, the decrease in phosphatidylcholine made lipsome formation and encapsulating E.coli more difficult.

In summary, we successfully prepared lipsome vesicles and encapsulate E.coli in lipsomes. And we could improve our method to further increase lipsome stability and encapsulation efficiency for E. coli.

Fig.13 (a)Optical microscope result of lipsome hydrated with water, PC:CH=7:2.(b)(c)Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:2.(d)Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:4.(e)Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:6.