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Team:OUC-China/Project Result
From 2014.igem.org
Result
Overview
Throughout the course of iGEM, we have carried out many experiments about our project. Now we have aquired a large amount of results and data.
Our experimental results are presented in two parts: one is the test ofthe double plasmid system, the other is the test of transfection effect.
We are proud that we have gained reasonable results and data to prove the feasibility of our project.
Mini plasmid & double plasmid system
mini plasmid
We get the oriT region of plasmid RP4 by means of PCR, and ligate it with the reporter gene BBa_J04450, then a non-self-controllable conjugated part is constructed.
In the meantime, we ligate BBa_J04450 with BBa_J01003 to test the conjugated ability of BBa_J01003.
Deactivate the OriTRP4
We attempt to deactivate the OriT of plasmid RP4 by homologous recombination, unfortunately, the first experiment failed. And we have no time to do experiment before the deadline.But, it is theoretical to deactivate the OriT. We will continue our experiment after the competition.
We make two plasmids enter in E.coli HB101, one is plasmid RP4 (the oriT site was deactivated) and another is mini plasmid carrying oriTRP4-RFP or oriTR-RFP. Firstly, oriTRP4-RFP and oriTR-RFP transfer in competent cells HB101 (by inducing artificially, from Qiu Zhigang) respectively. Secondly, by means of conjugation, we make the plasmid RP4 get in HB101 which mentioned above and already has a mini plasmid. Thirdly, we screen cells with two plasmids by chloramphenicol and kanamycin. Eventually, we acquire HB101 strains carrying two plasmids (RP4, oriTRP4-RFP or oriTR-RFP).
Conjugation
Test1. Conjugation between E.coli HB101 and Top10
We did conjugation experiment between HB101 and Top10 to test the conjugation ability of OriTRP4 (BBa_K1439000). We got a special strain from other lab which is sensitive to Streptomycin, while Top10 has streptomycin resistance. In order to test BBa_K1439000 better, we ligated OriTRP4 with the report gene RFP. After conjugation, we picked the red colonies by LB medium with resistance.
We designed a orthogonal experiment, it is showed in the table. The first column shows the strains used in coating, and the first row shows the antibiotics added in medium. Besides, the table shows the result ideally. The second row proves that HB101, plasmid RP4 and the backbone pSB1C3 don’t contain streptomycin resistant gene. The third row proves that Top10 is sensitive to chloramphenicol. And the forth row proves that Top10 can receive the mini plasmid after conjugation.
Conjugation between E.coli HB101 and Top10
| Conjugation (HB101 and Top10 mixed system) | Top10(no plasmid) | HB101(double plasmids system) | |
|---|---|---|---|
| Streptomycin | White colony | White colony | No colony |
| chloramphenicol | Red colony | No colony | Red colony |
| chloramphenicol & streptomycin | Red colony | No colony | No colony |
Table.1 We constructed a new part BBa_K1439001 to test the conjugation result. BBa_K1439001 is used in conjugation experiments below.
Result
Figure 1.
Figure 2.
Figure 1. is the result of conjugation experiment, but it doesn’t correspond to Table.1. Red fluorescent proteins are not expressed. So we picked the conjugated colonies and cultured. After culturing hours, we extracted the plasmid and sequenced. (Figure 2.)Finally, we verified that BBa_K1439000 could transfer in recipient cells with the help of plasmid RP4.
Test2. Conjugation between E.coil HB101 and Vibrio harveyi
We also test the conjugation ability of double plasmids system between E.coil HB101 and Vibrio harveyi.
The Vibrio harveyi is used as recipient cell and can be screened by Thiosulfate citrate bile salts sucrose agar culture medium (TCBS) with chloramphenicol.
Figure 3.
BBa_K1439001 can conjugate with Vibrio harveyi. The experiment details can be referred on protocol
At the same time we test the BBa_J01003 the conjugation ability by the same way.
Conjugation between E.coli HB101 and Top10
| Streptomycin | chloramphenicol | chloramphenicol & streptomycin | |
|---|---|---|---|
| Conjugation (HB101 and Top10 mixed system) | White colony | Red colony | Red colony |
| Top10(no plasmid) | White colony | No colony | No colony |
| HB101(double plasmids system) | No colony | Red colony | No colony |
Table
Table.2 We constructed a new part BBa_K1439002 which linked BBa_J01003 with the reporter gene BBa_J04450 to test the conjugation ability of BBa_J01003.
Result
Figure 4.
Figure 5.
From lane2 to lane5, they show the result of extracting plasmid from Top10 after conjugation. Lane1 shows the marker DL5000.
Figure 4 is the result of conjugation experiment, and it correspond to Table2.. In the mean time we picked the conjugated colonies and cultured. After culturing hours, we extracted the plasmid and sequenced. (Figure 5.) Finally, we verified that BBa_K1439002 could transfer in recipient cells with the help of plasmid RP4.
Conjugation between E.coil HB101 and Vibrio harveyi
We also test the conjugation ability between E.coil HB101 and Vibrio harveyi.
The Vibrio harveyi is used as recipient cell and can be screened by Thiosulfate citrate bile salts sucrose agar culture medium (TCBS) with chloramphenicol
Result
Figure 6.
BBa_K1439002 can conjugate with Vibrio harveyi. The experiment details can be referred on protocol.
TAT:H4 bind plasmid
We got the TAT:H4 protein. The protein TAT:H4 is a very important device in our program. It can bind plasmids, protect plasmids and transfer plasmids into cells. We performed gel retardation experiments. A delay in the electrophoretic mobility of the sample was observed and the level of which is coincident with increased concentration of TAT:H4. When the TAT:H4/DNA mass ratio reached 8:1, the gel mobility reached the lowest. It indicated that the protein TAT:H4 had the ability to form DNA-protein complex.
Figure 7.
From Lane 1 to 6. TAT:H4/DNA mass ratio reached 8:1, 6:1, 4:1, 2:1, 1:1, 0:1. And lane 7 is Marker DL5000.
TAT:H4 protect DNA
We used DNase to deal with the protein-DNA complex. Compared to the control, we could find that the DNA is not degraded by the DNase. We can draw a conclusion that the protein TAT:H4 can protect DNA from degradation by DNase, which provides precondition for transfection in vitro experiment.
Figure 8.
From Lane 1 to 6. TAT:H4/DNA mass ratio reached 8:1, 6:1, 4:1, 2:1, 1:1, 0:1. Lane 7 is pcDNA3.1(+)-EGFP and lane 8 is Marker DL5000.
Transfection
We put pcDNA3.1(+)-EGFP which bound with TAT:H4 into the cell-culture medium. After 24-48h, we hoped the green fluorescence appeared in the cells when the section of material was observed by fluorescent microscope with blue light. That means plasmid pcDNA3.1(+)-EGFP was absorbed by the cells, and the number of glowing green fluorescent cells outnumbered the control groups by a lot. However, we couldn't keep the concentrator asepsis and cell-culture contamination caused cells death. So we can't get prefect results in cell-culture transfection.
RT-PCR
Then we did the experiments on zebra fish. We injected 10uL protein-DNA complex into zebra fish. After 48h, we extracted total RNA from the tissue where we injected, and we verified that there existed RNA of our reporter gene, which means our transfection device is effective in zebra fish.
Figure 9.
图片描述
Figure 10.
The different EGFP's mRNA expression between injecting plasmid only and TAT-H4-plasmid. The efficiency of transfection of injecting TAT-H4-plasmid is higher 92.41% than injecting plasmid only at least.
We let our protein bind with pcDNA3.1-EGFP and then injected it into zebrafish’s muscle tissue .We injected 100ng plasmid-protein complexes into each experimental group fish and as for the control group we only injected 100ng plasmid per fish. There are 6 experimental groups and 6 control groups. Then we carried out RT-PCR experiments after 24h. It can be found that both of the experimental group and the control group fish had the transcription of mRNA but the experimental group’s transfection efficiency is higher than the control group. We did some quantitative analysis experiments and discovered this fact.
Lysis Device
After testing the device A for the first time, we got data that showed not remarkable but clear difference of the absorbance at 600nm among experimental group with a range of concentrations of arabinose. The data is shown below.
| Concentration of L-arabinose | Time after induction | ||
| 3 hours | 6 hours | 9 hours | |
| 1μM | 0.322 | 0.745 | 0.901 |
| 3μM | 0.247 | 0.741 | 0.927 |
| 6μM | 0.209 | 0.733 | 0.891 |
| 10μM | 0.191 | 0.750 | 0.924 |
| 100μM | 0.183 | 0.721 | 0.848 |
| 1M | 0.222 | 0.548 | 0.687 |
| 10mM | 0.132 | 0.631 | 0.685 |
But the other three devices had not yet been confirmed to work well before wiki freezed. It may be caused by homologous recombination, experimental details and other factors. We are trying to find out the exact reason and will manage to make difference to the devices by the giant jamboree.
