Team:HZAU-China/Labnotes

Parts connection and standardization:

Author: Zheng Yihui

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

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.

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 6.

Figure 6. 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 7

Figure 7. 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 8 and Figure 9 were exactly accord to our expectation which indicated that we constructed it successfully.

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Figure 8. 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 9. result of fluorescence intensity