Team:HZAU-China/Labnotes

Molecule Labnotes

Author: Yihui Zheng

Members: Yihui Zheng, Yajing Zhang, Zhixiang Liu, Xudong Luo, Xiaoxue Guo, Lu Wang, Siqi Li.

Introduction:

We have eight gene circuits to construct (as shown in the Figure 1), and after finishing these circuits, we will integrate some of them into one plasmid to form two repressilators and two detection devices (as shown in the Figure 2a and Figure 2b).

Figure 1. eight gene circuits

Figure 2a. first rewirable circuit and its detection device

Figure 2b. second rewirable circuit and its detection device (quorum sensing)

May

Content: we found the parts we needed in the Plate Kits and did a lot of transformations to get the plasmid for subsequent experiments.

Parts list: BBa_J06602 BBa_E0420 BBa_K886000 BBa_R1051 BBa_P0412 BBa_R0040 BBa_P0451 BBa_R0011 BBa_R0051 BBa_I714075 BBa_J61047 BBa_R0063 BBa_K516022 BBa_K805016 BBa_J37032 BBa_K081019 BBa_K773003 BBa_I13602 BBa_R0061 BBa_R0062 BBa_R0063 BBa_C0060 BBa_B0015 BBa_I718016 BBa_I0462 BBa_P0440 BBa_E0022 BBa_J01010

June

Content: eight gene circuits’ construction

Construction details:

1.Construction of gene circuit one: we used standard method of biobrick connection to put the coding sequence of cI protein behind P_LlacI promoter. But we failed to construct it because of some unknown reasons. The sequence results showed that there is either a pretty long and unknown segment which insert into the coding sequence of cI protein or deficiency of the sequence of P_LlacI promoter(as shown in the Figure 3). So, we had an assumption that there were little LacI proteins in DH-5α, and it was not much enough to repress P_LlacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host.

Figure 3. diagram of sequence results

2.Construction of gene circuit three: Firstly, we used standard method of biobrick connection to put the coding sequence of LacI protein behind lox66 because the length of lox66 was 34bp and it was too small to purify from the enzyme-digested product and then put it in front of LacI protein. Due to the same reason, we put the product of previous step behind P_Ltet01 promoter to finish this gene circuit. Compared to construction of other gene circuits, it seemed that this gene circuit was the simplest job we’ve made since that we only tried once and took almost eight days to finish this work.

3.Construction of gene circuit two: we constructed this gene circuit by following the method we’ve mentioned above. The only difference from the construction of gene circuit three is that we put the coding sequence of TetR protein behind lox71. By the way, lox66 and lox71 are two recombination sites which are recognized specifically by Cre protein. To our surprise, when we did double-digest to this circuit for verification, we found that the length of product of enzyme-digested was not right and there was an extra stripe that we didn’t know what it was. Results are shown in the Figure 4. According to the results, we had another assumption to explain the phenomenon: there were some recombination systems in DH-5α, resulting in homologous recombination of two strains which had synclastic lox71 site. Definitely, We’ve done a lot of work to verify our assumption which will be explained in the part of characterization.

Figure 4. the stripes which arrows point towards are enzyme-digested product of gene circuit two and the rightest stripe in each figure is DL-5000 marker. There were at least three stripes and the length of extra stripe is wrong.

July

Content: eight gene circuits’ construction

1.Construction of gene circuit four: we used standard method of biobrick connection to put the coding sequence of mCherry protein behind P_λ promoter. And when we planned to construct the part CFP expression controlled by P_LlacI promoter, we didn’t find CFP with LVA tag in the Plate Kits, so we decided to get this part through PCR which means that we added a LVA tag behind the coding sequence of CFP. And then, we used standard method of biobrick to connect P_LlacI promoter with the coding sequence of CFP to finish the whole circuit. Figure 5 will show the visual effects of mCherry protein expression and CFP expression under the fluorescent microscope.

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Figure 5. a was without any excitation light; b and c were mCherry protein expression and CFP expression under the fluorescent microscope

2.Construction of gene circuit five: Because of the complex stem-loop structure of RNA aptamer, it was very hard to get this part by overlapping PCR. So, we found a company to help us to synthetize the sequence of RNA aptamer, and we standardized RNA aptamer and also we connected it with the promoter (lambda cI regulated). To test whether it works or not, we synthetized corresponding fluorophore called DHMBI, and saw the figure under the fluorescent microscope as shown in the output module.

3.Construction of gene circuit six and eight: Construction of gene circuit six and eight: These two gene circuits are familiar in their structure and then, we constructed them simultaneously. Firstly, we divided the circuit into two parts: riboregulator and Cre protein with double terminators. When it came to the synthesis of riboregulator, we designed four specific primers which had almost 20 overlapping sequence and conducted twice PCR to get our riboregulator (more details are shown in our protocol and the input module). Then, we connected both of them by the standard method of biobrick connection. Figure 6a and 6b are the visual effects of gene circuit six without AHL and gene circuit with AHL under the fluorescent microscope, and Figure 6c is the quantitative result of these two samples and we can find that there is a obvious difference between gene circuit six without AHL and with AHL which is accord to our expectation.

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Figure 6. a and b are the visual effects of gene circuit six without AHL and with AHL; c is the quantitative result of these two samples

August

Content: we verified our first assumption that the overexpression of cI protein may restrain the growth of host which cause the failure of construction of gene circuit one, so, we designed another method to construct it and finally made it. Then, we integrated three circuits into one plasmid to form our repressilator.

1.Verification for the first assumption that there were little LacI proteins in DH-5α, and it was not enough to repress P_LlacI promoter, resulting in the overexpression of cI protein which may restrain the growth of host:

We designed a primer contained PstI site which could match the middle part of the coding sequence of cI protein, and then, we conducted a PCR by using VF2 primer and the designed primer to get a sequence which can express an inactive cI protein. We put this sequence behind P_LlacI promoter through the same way mentioned above and the sequencing results showed that we constructed it and it proved our assumption to some extent. Then, we came up with a different way to construct our first repressilator which will be explained in the following.

2.New way to construct our first repressilator: Due to toxicity of cI protein on host bacteria and the expression of cI protein was regulated by P_LlacI promoter, we came up with a new method that putting the gene circuit three in front of P_LlacI promoter because circuit three would express LacI protein to repress the activation of P_LlacI promoter and thus, cI protein would not be overexpressed. So, we digested circuit three by EcoRI enzyme and SpeI enzyme and digested P_LlacI promoter by EcoRI enzyme and XbaI enzyme, and then do a ligation to get a circuit contained P_LlacI promoter controlled by LacI protein. After that, we connected the coding sequence of cI protein with the previous product to get a compound contained circuit one and circuit three. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 7.

Figure 7. plasmid contained circuit one and circuit three (a,b)

September

Content: we constructed the first repressilator and change the vector of detection device from high copy to low copy.

1.The construction of first repressilator: The first repressilator contained three gene circuits: circuit one, two and three, and at that moment, we connected circuit one with circuit three, lacking of circuit two. We tried our best to construct it and due to the special condition that we needed to connect this two part reversely and change the vector of repressilator from high copy to low copy, we designed a new method to connect two parts reversely that we digested circuit one and three by EcoRI enzyme and PstI enzyme, meanwhile, we digested circuit two by EcoRI enzyme and XbaI enzyme and target vector by XbaI enzyme and PstI enzyme, leading to the result that there was a common EcoRI site for ligation between circuit two and the compound contained circuit one and three. We’ve tried at least five times in this construction and we tried to separate this procedure into two steps: connection first and then change the vector, but all tries failed. Surprisingly, in the sixth trial, we may design an optimal system of ligation including the proportion between target DNA and vector, we constructed this repressilator successfully. Gel electrophoresis spectrum diagram of plasmid is shown in Figure 8

Figure 8. plasmid of repressilator

2.Changing the vector of detection device from high copy to low copy: we should change the vectors of two detection devices including the compound contained circuit four and circuit six and the gene circuit eight, and at the same time, we changed the vector of RNA aptamer generator which played the same function as fluorescent protein. The indicator to judge whether it had changed successfully or not was the resistance gene: the vector of high copy had chloramphenicol-resistance gene and the vector of low copy had Ampicillin-resistance gene.

October

Content: we constructed our seventh gene circuit about quorum sensing and tested the intensity of GFP through fluorescent microscope and microplate reader.

Construction of gene circuit seven: We divided this circuit into four parts: AiiA protein generator, LuxI protein generator, LuxR protein generator and GFP generator. Both the parts GFP expression controlled by LuxR promoter and luxR protein generator under regulation were found in Plate Kits. As for another two parts, we did them the same thing as the construction of gene circuit three. And then, we did the same thing as the construction of our first repressilator to connect three segments to form our seventh gene circuit. To judge whether it was connected correctly and successfully, we co-transformed our gene circuit seven and eight (with Cre protein) and transformed only the gene circuit seven (without Cre protein) as a comparison, the results shown in the Figure 9 and Figure 10 were exactly accord to our expectation which indicated that we constructed it successfully.

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Figure 9. a was without any excitation light; b was the phenomenon when we co-transformed the gene circuit seven and eight (with Cre protein); c was the phenomenon when we only transformed the gene circuit seven (without Cre protein) as a comparison.

Figure 10. result of fluorescence intensity

Cellular Labnotes

1. Basic works

1.1 Confirmation of the unexpected recombination of lox71 site

Author：Lu Wang

1.1.1 Experimental Background

Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP site which can also be specifically recognized by Cre recombinase. Our purpose is to confirm that unexpected recombination of lox71 site exits through sequencing result and observation under fluorescence microscope.

1.1.2 Materials and equipment

Materials:
Part: Lac Promoter; Lox71 site (BBa_K886000); mCherry (BBa_J06504)
Strain: Escherichia. Coli (DH5α, DH10b, BL21)
Glycerinum, LB broth and plate, assorted antibiotics
Equipment:
Fluorescence microscope

1.1.3 Method

Device Assembly
Ligate the Lac promoter with lox71 site, and lox71 site with mCherry using the Bio-brick assembly.

Fig.1

Fig.2

Change the sequence of bio-brick prefix to make it recognized by Pst I restriction endonuclease utilizing a PCR reaction.

Obtain the PCR product:

Fig.3

Join the Lac promoter-lox71 and lox71-mCherry fragments with double digestion and ligation. Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.

Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.

Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.

Testing
(Whether the recombinase with low specificity expressed by E.coli endogenously could catalyze a recombination process between a pair of lox71 sites)

Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.

Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.

Confirmation
(Confirm the plasmids that code Cre recombinase from the registry work properly in E.coli)

Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.

Fig.4

Fig.5

1.1.4 Experimental Results and Discussion

Observation on Group Level

Fig.6

The results present that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.

Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.

Fig.7

Observation on Individual Level

The individual cell of DH5α and DH10b were observed through the fluorescence microscope, and the photographs are shown below.

Fig.8 (Strain DH5α, 10*40)

Fig.9 (Strain DH10b, oil immersion lens, 10*100, white light)

Fig.10 (StrainDH10b, oil immersion lens, 10*100, UV-light, red filter)

Fig.11 (Strain DH5α, oil immersion lens, 10*100, white light)

Fig.12 (StrainDH10b, oil immersion lens, 10*100, UV-light, red filter)

The recombinase with low specificity expressed by E.coli endogenously could catalyze a recombination process between a pair of lox71 sites, but with an extremely small probability. The rate of recombination between the lox71 sites in DH10b is lower than that in DH5α.

Sequencing

We sequenced the plasmids of the positive clones and the result was not bimodal. So far, it could be said that lox71 sites can recombine under the catalysis of the non-specific Recombinase of E.coli, but the probability is so small that neither the construction of plasmids nor the following experiments will be affected.

Discussion

The recombinase with low specificity expressed by E.coli endogenously could catalyze a recombination process between a pair of lox71 sites, but with an extremely small probability.

The rate of recombination between the lox71 sites in DH10b is lower than that in DH5α.

Recombination process between a pair of lox71 sites will be catalyzed by E.coli endogenous recombinase, but the probability is small enough that neither the construction of plasmids nor the following experiments will be affected.

The probability of unexpected lox71 site-based recombination in DH10b is smaller than that in DH5α.

The plasmids that code Cre recombinase from the registry work properly.

1.2 Promoter test

Author: Yuan Lu

1.2.1 Experimental Background

The three devices used in our project are concerned with plenty of functions such as oscillation, feedback and feedforward. Since the difference between frequencies of initiation of the promoters has critical impact on the functions of these regulatory devices, it is important to measure the relative frequencies of initiation between these promoters.

Our purpose is to measure the relative initiation frequencies of promoters used in our devices, which are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011.

1.2.2 Materials and equipment

Materials
Parts: Promoters (BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063、BBa_R0011), mCherry (BBa_J06504), RBS (BBa_B0034), Terminator (BBa_B0015)
Strain: Escherichia. Coli (DH5α)
LB broth and plate, Assorted antibiotics

Equipment
Multi-function Enzyme-labeled instrument

1.2.3 Method

Transformation of DH5α with each of the plasmid necessary in our projects, incubation on a accordingly selective plate for 14 hours or so at 37 and then cultivate the single colony in LB broth for 8 hours at 37 Celsius degree. Extracting plasmid from bacteria and after the sequencing result shows the correct sequence, stored the plasmid at -20 Celsius degree.

Construction of the vectors of different promoters as illustrated by biobrick method. Then after the sequencing prove the correction of 6 vectors constructed transformed them and other 6 same vectors but without promoters upstream respectively to the competent cell DH5α in 12 different tubes, and then cultured at 37 Celsius degree.

After culturing for 12 hours measuring the fluorescent degree of 12 different bacteria (6 in which without vector with promoter serve as control) by enzyme-labeled instrument.

To determine the effect of inducer, IPTG was added to each of tube in which bacteria contains vector with BBa_R0040 or BBa_R0011 and its according control and then culturing for another 12 hours and then measuring the fluorescent degree by enzyme-labeled instrument.

1.2.4 Experimental Results and Discussion

Fig.13 Detection of the part BBa_R0040 after adding inducer

Fig.14 The detection of the relative strength of the promoters

Analysis:

Fig.15 Detection of the part BBa_R0040 after adding inducer

Fig.16 The detection of the relative strength of the promoters

Based on the data from Fig.13, we do the statistical analysis of the detection of the part BBa_R0040 after adding inducer. With analysis diagram shows in Fig.15, the inductive effect of the inducer is not evident in low concentration. However, inductive effect of the inducer is evident when the concentration is up to 10µM.

Based on the data from Fig.14, we do the statistical analysis. With analysis diagram shows in Fig.16, we can conclude that the rank of relative strength of the promoters from strong to weak is: BBa_R0063、BBa_R0011、BBa_J23119、BBa_R1051、BBa_R0040、BBa_R0062.

1.3 pCusC, PpcoA sequencing and promoter test

Author: Liu Tian

1.3.1 Experimental Background

iGEM encourage the communication between teams and help others. So, when we heard HUST-China has something wrong about their promoter, we decided to give a hand. The one problem they had is they tried many times but failed to sequencing those promoters in pET28a, and the other is they put mRFP under the promoter to do a promoter test but didn’t get result. So we got A&B promoters with mRFP from them.

1.3.2 Proposes

pCusC & PpcoA promoters sequencing
pCusC & PpcoA promoters test

1.3.3 Materials and equipment

Materials:
pCusC & PpcoA promoters with mRFP in pET28a plasmid
LB medium
Kanamycin

Equipment:

Multifunctional microplate reader
Shaking incubator

1.3.4 Method

Sequencing

We design the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequencing the promoters. And Tsing Ke Biology Technology Company help us synthetize the primer and sequencing those plasmid. You can get the sequencing results of two samples at supplement 1. Analyzed the sample pCusC’s sequencing result, we find that the T7 promoter has not be digested , there is no pCusC in the plasmid, but mRFP has successful ligated. So we can observe the red is the red fluorescent protein expression by the T7 promoter. Therefore, in the subsequent promoter test, we abandoned the pCusC samples. In the PpcoA samples we successfully found PpcoA and mRFP. However, at the end of PpcoA‘ssequencing result, there is more 6bp than HUST’s sequence. We take PpcoA promoter to detection.

Promoter test

Add 2mL LB culture medium, 2uL Kanamycin (50mg/L) and 20uL bacterial samples to a 5mL centrifuge tube. Shaken overnight in the 37°C shaking incubator and set the rotational speed at 180 rpm/min.

Add CuSO4 solution to induce in concentration of 0, 0.02, 0.1 and 1mM. Set three copies for each concentration as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.

Add 200uL bacterial samples from each centrifuge tube to the 96-well plate. Set two copies for each centrifuge tub as repetitions. Meanwhile, add LB culture medium containing and not containing 1.0mM CuSO4 as blank controls.

Read the OD600 and fluorescent intensity (with the emission wavelength at 607nM and excitation wavelength at 584nM) using a multifunctional microplate reader.

Divided the fluorescent intensity result using the value of OD600. Record the data for compare and analysis.

1.3.5 Result and discussion

Fig.17

When we add the 1.0mM Cu to bacterium solution，we observed the color change of the visible to the naked eye. So we try to test the 1.0mM Cu in LB medium. Then we find its fluorescence value and the value of OD600 with only LB samples have a large difference. So we decided to use the 1.0mM Cu in LB medium as the blank samples of the 1.0mM control.We can observe from the figure, when the copper ion concentration is range of 0-0.1mM, with the increase of concentration of copper ion, promoter strength increased. In the copper ion concentration of 1mM, the promoter strength decreased. To this end, we intend to in the copper ion concentration in the range of 0.1-1mM is provided with a plurality of gradient measured next.

2. Input module test

2.1 Cre recombinase function test

2.1.1 Experimental Background

To confirm the function of Cre recombinase

2.1.2 Materials and equipment

Materials： Part：mCherry (BBa_J06504)
Strain: Escherichia. Coli(DH5α, DH10b, BL21)
LB broth and plate, assorted antibiotics

Equipment：
fluorescence microscope

2.1.3 Method

Test whether the recombinase with low specificity expressed by E.coli endogenously could catalyze a recombination process between a pair of lox71 sites.

Transform the confirmation device into DH5α, DH10b, and BL21, and pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol and IPTG inducer.

Observe the culture’s color and take a small amount under the fluorescence microscope to observe the single bacteria. Extract the plasmids from single colonies and sequence the plasmid with general purpose primer.

Confirm the plasmids that code Cre recombinase from the registry work properly in E.coli.

Co-transform the confirmation device and plasmids that code Cre recombinase into DH5α, pick the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observe the culture’s color.

Fig.18

Fig.19

2.1.4 Result and discussion

Fig.20

Fig.21

The results present that the culture of Negative Control and Group One has become opaque but still keep being pale yellow. The Group Two’s culture has turned red obviously and also opaque.

Therefore, conclusion could be drawn that plasmids which code Cre recombinase in the registry work properly and the confirmation device we assembled is valid.

Observation on Individual Level

The individual cell of DH5α and DH10b were observed through the fluorescence microscope, and the photographs are shown below.

Fig.22

Fig.23

Fig.24

Fig.25

Fig.26

2.2 Riboregulator function test

Author：Lu Wang

2.2.1 Experimental Background

We want to know under the participation of taRNA and crRNA, whether can reduce leakage of Plac expression.

2.2.2 Materials and equipment

Material：
Strain: Escherichia. Coli(DH5α) LB broth and plate, Assorted antibiotics

2.2.3 Method

Fig.27

Fig.28

Fig.29

First, built the three devices and from top to bottom Numbers for 1 2 3, and then, respectively transform the three plasmid into BL21 competent cell.Pick monoclonal colony.Add 5ml LB liquid and incubate at 37 Celsius degree for 8 hours. Secondly, add 5ul of LB liquid cultural medium from each tube to two new tubes named A, B respectively with 5ml M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubating at 37 Celsius degree for 4 hours. Lastly, measuring the fluorescent degree by utilizing the enzyme-labeled instrument.

2.2.4 Result and discussion

2.3 Tight regulation test to input module

Author: Lu Wang

2.3.1 Experimental Background

Since the high catalytic capability of the Cre recombinase, a small amount of recombinase can lead to the recombination between the lox sites, combine with the basal expression of the operon in our input device, the lox sites in our others device can easily be recombined in the bacteria before the stimulation by the inducer (IPTG in this case) on the repressor (lacI in this case). To address this problem, we add a riboswitch as a second layer of control upstream to the ORF of the cre to suppress the translation of the mRNA of the reconbinase in absence of the inducer. We wish to tightly control the expression of the Cre recombinase in both transcription and translation. Thus, in our input system, only stimulation by adding inducer can the expression of the recombinase be activated, while the production of the Cre can be extremely low in absence of the inducer. We undertook the experiment to verify the function of this device.

2.3.2 Materials and equipment

Material:
Vector-pSB1A3,pSB1C3
Strain: Escherichia. Coli (DH5α)
LB broth and plate, Assorted antibiotics

Equipment：
Multifunctional microplate reader

2.3.3 Method

Co-transformation of bl21 with input device and verification device and plate the transformation onto a selective plate, incubate at 37 Celsius degree for 14hours or so.

Incubate 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours.

Add 5ul of LB liquid cultural medium from each tube to two new tubes named A, B respectively with M9 broth in it. Add 0.1ul IPTG to all A tubes (1A, 2A, 3A) while add nothing to all B tubes (1B, 2B, 3B). Incubating at 37 Celsius degree for 4 hours.

Measuring the fluorescent degree by utilizing the enzyme-labeled instrument.

Prediction and analysis:

If the fluorescent degree in the cultural tube with IPTG showed much less intensity than in the tube without it, then it indicated that the device had achieved its function for reducing cre translation as expected.

If the fluorescent degree in the cultural tube with IPTG showed almost same or even larger intensity than in the tube without it, it meant the devices had failed its purpose. While 3 reasons may be responsible for its failure. The first reason may be the failed function of riboswitch to repress the translation process. Second, despite the strong repression of translation due to the riboswitch, the tremendous catalytic activity of cre complements the low concentration of cre in bacteria. Third, the low production of lacI in bl21 bacteria is unable to repress the operon efficiently which leads to the high expression of both taRNA and cre mRNA and thus undermine the function of riboswitch.

2.3.4 Result and discussion

Fig.30

Fig.31

As showed above, even though the addition of riboswitch does reduce a little of the production of Cre protein, the level of reduction is too slight and can hardly exert some critical influence on the experiment.

3. Output module test

3.1 The Detection of Aptamer (Qualitative Detection)

Author: Yajing Zhang, Siqi Li

3.1.1 Experimental Background

Detect the aptamers whether available individually. In the case of the entire project is out of the entire project.

3.1.2 Materials and equipment

Material:
DMHBI, M9 broth, E.coli(DH5α)

Equipment:
fluorescence microscope

3.1.3 Method

The synthesis of DMHBI

N-Acetylglycine (5.26 g, 0.045mol), anhydrous sodium acetate (3.69 g, 0.045mol), 4-hydroxy-2,3-dimethoxybenzaldehyde (8.19 g, 0.045mol), and acetic anhydride (15 ml) were stirred at 90℃for 2 h. After allowing the reaction to cool to room temperature, cold ethanol (20 ml) was added while still stirring and the reaction was left stirring overnight at 4℃. The resulting crystalline solid was then washed with coldethanol, hot water, hexanesand dried to afford 9.65 g (yield 70%) of 3as a pale yellow solid: H NMR (500 MHz,CDCl_3) δ7.56 (s, 2H), 7.18 (s, 1H), 3.98 (s, 6H), 2.54(s, 3H), 2.39(s, 3H); LC/MS (LC: gradient 20-95% MeCN [0.1% HCO_2 H] over 2.5min, 0.5 ml/min flow rate, MS: ESI^+): retention time, 2.15 min; purity, 95%; 306.36 〖[M+H]〗^+. Then we can get Compound 3.Compound 3 (1.12 g, 0.005mol) was refluxed with 15 ml of ethanol, 1 ml of 40% aqueous methylamine, and 700 mg of potassium carbonate for 4h. The reaction mixture was removed from heat and upon cooling formed an orange precipitate. The precipitate containing the product was filtered and washed briefly with cold ethanol. The precipitate was then redissolved in a 1:1 mixture of ethyl acetate and 500 mM sodium acetate pH 3.0. The organic layer was separated, dried with anhydrous sodium sulfate and solvent was removed under reduced pressure to yield 717 mg (yield 52%) of DMHBI as an orange solid: 1H NMR (500MHz, DMSO-d_6) δ7.62 (s, 2H), 6.90 (s, 1H), 3.80 (s, 6H), 3.09 (s, 3H), 2.34 (s, 3H). The process during synthesis has been shown in Fig.28.

Fig.32

Then the DMHBI should be stored in such condition:
Avoid light vacuum
Preserve it in low temperature
Isolate it from oxygen

According to the literature, we will get an orange solid. But in reality, we get something dark brown. After detection, our products are in liquid state and we get 40% in purity of product. In order to get more product in high purity, we invite ShaoQing Zhuang who is a student in Huazhong University of Science and Technology to help us with the synthesis of the DMHBI. And finally we get more than 95% in purity of product.

Inoculation and training E.coli all the night. When the =0.8, take out and centrifugal 1.5ml bacterial suspension(6000rpm,1min), and resuspend it with 1ml M9 medium. Then training it 1h in the shakers(37℃, 170rpm). Remove the background of bacterial. Resuspend it with 1ml M9 medium, and add 1μl DMHBI(MW=276). After being dissolved, the concentration of DMHBI is 200mM, And the concentration of what we needed is 200μΜ. Mix and training it 10min in the shakers(37℃, 170rpm). Detect it by fluorescence microscope.

3.1.4 Result and discussion

The result of detect E.coli with promoter and aptamer by blue exciting light is green point. And the result of detect E.coli with promoter and aptamer by purple exciting light is blue point. Hence, the result is same with the reference.

Fig33. The result of detect E.coli with promoter and aptamer by blue exciting light is green point. (10*40, blue filter).

On the one hand, the display of fluorescence microscope is a plane picture. Hence, there are different picture, and we got different fluorescence point, with different focal length. The light, sent from aptamer, is similar to the light sent from GFP, so the light’s penetrating power is weak. Hence, not any bacterial to fluoresce, and we can not see anyone to fluoresce, and we can not see a piece of light. We can see fewer solitary light point.

On the other hand, the glowing effect depends on the incubation time of E.coli with DMHBI, and it could be found in the result of the detection of the same day and the detection of the next day. But there are another problem with the incubation time extend. The bad growth state of E.coli will affect the observation.

We want take a photo that distinct and have dense fluorescence point. But, there are no ideal picture.We absent E.coli with promoter and no DMHBI. And we do the comparison of other sample in the gel image system ,we can see the result as follow. The sample of intermediate is E.coli with promoter and aptamer,and it is the brightest. The sample of downmost is E.coli with promoter , and it is brighter than the sample of uppermost . The sample of uppermost is control test sample, only with M9 medium and DMHBI ,and it is the darkest.

Fig.34 The picture of reference.

Fig.35 The picture of test.

3.2 Fluorescent proteins with LVA tag

3.2.1 Experimental Background

Testing whether Fluorescent proteins with LVA tag can degrade rapidly or not for further use in our project.

3.2.2 Materials and equipment

Material:
DMHBI, M9 broth, E.coli (DH5α)

Equipment:
fluorescence microscope

3.2.3 Method

Fig.36 output module of plan 2

3.2.4 Result and discussion

Fig.37

4. Processing module test

Reference：

Paige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.