Difference between revisions of "Part:BBa K1439000:Experience"

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__NOTOC__
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==Test1. Conjugation between E.coli HB101 and Top10==
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===Test1. Conjugation between E.coli HB101 and Top10===
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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.
 
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.
  
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===Applications of BBa_K1439000===
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Conjugation between E.coli HB101 and Top10
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We have test the oriTRP4 BBa_K1439000 in a conjugation system in which pSB1C3 with the help of plasmid RP4, and ''E. coli'' HB101 as donor and'' E.coil'' Top10 as recipient cells. We test the conjugative competence of oriTRP4 BBa_K1439000 by conjugate between HB101 and Top10. We use the RFP (BBa_J04450) marks the plasmids containing oriTRP4 BBa_K1439000. We get HB101 without streptomycin resistance form Zhigang Qiu. Top 10 has streptomycin resistance. After conjugation, we select recipient cells by streptomycin resistance LB medium and statistical number of red bacterial colony.
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{| class="wikitable"
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===User Reviews===
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<!-- DON'T DELETE --><partinfo>BBa_K1439000 StartReviews</partinfo>
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<!-- Template for a user review
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{|width='80%' style='border:1px solid gray'
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|width='10%'|
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!    !! Conjugation (HB101 and Top10 mixed system) !! Top10(no plasmid) !! HB101(double plasmids system)
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<partinfo>BBa_K1439000 AddReview number</partinfo>
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<I>Username</I>
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| Streptomycin || White colony || White colony || No colony
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Enter the review inofrmation here.
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| chloramphenicol || Red colony || No colony || Red colony
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|};
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<!-- End of the user review template -->
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| chloramphenicol & streptomycin || Red colony || No colony || No colony
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<!-- DON'T DELETE --><partinfo>BBa_K1439000 EndReviews</partinfo>
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|}
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Test
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Table.1 We constructed a new part BBa_K1439001 to test the conjugation result. BBa_K1439001 is used in conjugation experiments below.
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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.
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=== Result ===
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[[File:OUC-China_Parts_K1439000IMG1.jpeg]]
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Figure 1
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[[File:OUC-China_Parts_K1439000IMG2.jpeg]]
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Figure 2.
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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.
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===Experiment materials===
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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.
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Conjugation
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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.
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==Test2. Conjugation between E.coil HB101 and Vibrio harveyi==
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We also test the conjugation ability of double plasmids system between E.coil HB101 and Vibrio harveyi.
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The Vibrio harveyi is used as recipient cell and can be screened by Thiosulfate citrate bile salts sucrose agar culture medium (TCBS) with chloramphenicol.
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===Result===
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https://static.igem.org/mediawiki/2014/0/0c/OUC-China_result_conjugation_OriTRP4hujun.jpg
  
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Conjugate between ''E.coil'' HB101 and ''E.coil'' Top10.
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Figure 3
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BBa_K1439002 can conjugate with Vibrio harveyi. The experiment details can be referred on 2014OUC-China wiki.

Latest revision as of 02:07, 18 October 2014

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

OUC-China Parts K1439000IMG1.jpeg

Figure 1

OUC-China Parts K1439000IMG2.jpeg

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

OUC-China_result_conjugation_OriTRP4hujun.jpg

Figure 3 BBa_K1439002 can conjugate with Vibrio harveyi. The experiment details can be referred on 2014OUC-China wiki.