Part:BBa_K1439002:Experience
Contents
Test1. Conjugation between E.coli HB101 and Top10
Test1. Conjugation between E.coli HB101 and Top10 We do conjugation experiment between HB101 and Top10 to test the conjugation ability of OriTR (BBa_J01003). We got a special strain from other lab which is sensitive to Streptomycin, while Top10 has streptomycin resistance. In order to test BBa_J01003 better, we ligate OriTRP4 with the report gene RFP. After conjugation, we pick the red colonies by LB medium with resistance.
Conjugation between E.coli HB101 and Top10
| HB101(double plasmids system) | 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.1 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.
Result
Figure 1
Figure 2.
Figure 1.is the result of conjugation experiment, and it correspond to Table.1. In the mean time we picked the conjugated colonies and cultured. After culturing hours, we extracted the plasmid and sequenced.（ Figure 2.）From lane2 to lane5, they show the result of extracting plasmid from Top10 after conjugation. Lane1 shows the marker DL5000. Finally, we verified that BBa_K1439002 could transfer in recipient cells with the help of plasmid RP4.
Experiment materials
We use HB101 with BBa_K1439002 as donor cells, and use Top10 as recipient cells. Both of them were cultured 12hours in Luria-Bertrani (LB) broth. And the mediums we used are LB mediums with different antibiotics; they are chloramphenicol, streptomycin, both of chloramphenicol and streptomycin.
Conjugation
We mixed the cells at a donor: recipient ratio of 1:3. After 3 hours of static culture at 37℃ in incubator, we plated it on appropriate selective medium. The experiment details can be referred on 2014 OUC-China wiki.
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.
Result
Figure 3 shows that BBa_K1439002 can conjugate with Vibrio harveyi. The experiment details can be referred on 2014 OUC-China wiki.
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