Part:BBa_K1439000:Experience

Test1. Conjugation between E.coli HB101 and Top10

We do 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 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

	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.

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, 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. ） 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_K1439000 could transfer in recipient cells with the help of plasmid RP4.

Experiment materials

We use HB101 with BBa_K1439001 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 2014OUC-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 BBa_K1439002 can conjugate with Vibrio harveyi. The experiment details can be referred on 2014OUC-China wiki.