Team:LCG-UNAM-Mexico/AbrahamJurnal
From 2009.igem.org
(Difference between revisions)
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Use 10uL of this DNA extract as template for the PCR | Use 10uL of this DNA extract as template for the PCR | ||
- | The mixture | + | The mixture to perform the PCR is: |
- | + | 23uL H2O | |
- | + | 2.5uL Forward primer | |
- | + | 2.5uL Reverse primer | |
- | + | 3.5uL dNTPs | |
- | + | 2.5uL MgCl2 | |
- | + | 5uL Buffer10X | |
- | + | 10uL DNA template | |
- | + | 1uL taqDNA polymerase. | |
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Apply a heat shock at 41°C for 1 minute.<br> | Apply a heat shock at 41°C for 1 minute.<br> | ||
Incubate on ice for 5 minutes.<br> | Incubate on ice for 5 minutes.<br> | ||
- | Incorporate 1mL of liquid-LB medium.<br> | + | Incorporate and mix well 1mL of liquid-LB medium.<br> |
+ | Mix at 250RPM for 1 hour.<br> | ||
+ | Centrifugue to concentrate for 1 minute at 13000RPM. | ||
+ | Discard the supernatant. | ||
+ | Plate into a petri box. | ||
+ | |||
+ | |||
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RBS-RFP is going to be used after the asRNA-double_transcriptional_terminator as a reporter gene. (Check asRNA design) | RBS-RFP is going to be used after the asRNA-double_transcriptional_terminator as a reporter gene. (Check asRNA design) | ||
- | RBS-RFP-T.terminator If is the case that we can not afford the asRNA after other constructions are finished, we will use this biobrick as a reporter, It would be with a promoter inducible with luxR-AHL dimer (BBa_R1062) | + | RBS-RFP-T.terminator If is the case that we can not afford the asRNA after other constructions are finished, we will use this biobrick as a reporter, It would be with a promoter inducible with luxR-AHL dimer (BBa_R1062). |
+ | |||
+ | '''July 14th''' | ||
+ | ---- | ||
- | |||
Plan to ligate double terminators (BBa_B0015) at the end of the AHL making enzyme (BBa_C0261) the kanamycine resistance cassette (BBa_P1003) | Plan to ligate double terminators (BBa_B0015) at the end of the AHL making enzyme (BBa_C0261) the kanamycine resistance cassette (BBa_P1003) | ||
- | July 17-26th | + | '''July 17-26th ''' |
+ | ---- | ||
We MUST take vacation, the lab’s people will be on vacation and we’re not able to stay here without supervision of the advisor. We are supposed to read about general things involved in our project and also about applications in human’s health. | We MUST take vacation, the lab’s people will be on vacation and we’re not able to stay here without supervision of the advisor. We are supposed to read about general things involved in our project and also about applications in human’s health. | ||
+ | '''Aug 6th''' | ||
+ | ---- | ||
- | |||
We recieved an e-mail from Mr. Gene, They say that E3 and E9 colicin domains have always been tricking to synthesize, The E3 toxin has already been amplified by PCR, but the E9 sequence has been dificult to obtain. | We recieved an e-mail from Mr. Gene, They say that E3 and E9 colicin domains have always been tricking to synthesize, The E3 toxin has already been amplified by PCR, but the E9 sequence has been dificult to obtain. | ||
The GFP biobrick is cloned into any plasmid from Mr.Gene | The GFP biobrick is cloned into any plasmid from Mr.Gene | ||
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It seems that they were not able to clone neither E3 nor E9 colicines, we are worried about the dificulties that we will have to clone them. | It seems that they were not able to clone neither E3 nor E9 colicines, we are worried about the dificulties that we will have to clone them. | ||
+ | '''Aug 7th''' | ||
+ | ---- | ||
- | |||
From the transformations we performed on Aug 5th we observed some colonies, we expected them to grow in just 24hrs, but it took almost 2 days. we decided to perform colony PCR from. | From the transformations we performed on Aug 5th we observed some colonies, we expected them to grow in just 24hrs, but it took almost 2 days. we decided to perform colony PCR from. | ||
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As seen in the 1% Agarose gel, we got 4 true positive colonies in the OGR + BBa_B0015 transformation, and 2 true positive for BBa_J04450 + Cox. | As seen in the 1% Agarose gel, we got 4 true positive colonies in the OGR + BBa_B0015 transformation, and 2 true positive for BBa_J04450 + Cox. | ||
- | September 7th | + | '''September 7th''' |
+ | ---- | ||
We reasoned that the idea of asRNA. If we design and prove that is functional we will repress the fage infection easier. | We reasoned that the idea of asRNA. If we design and prove that is functional we will repress the fage infection easier. | ||
The idea is that infected bacteria will send AHL to alarm their neighbors about the infection, afterwards the bacteria close to this place will express the asRNA, so if lytic phase of phages is faster than the transcription machinery and the toxin’s action time (This implies that the first infected bacteria will “lose” even when contains the construction), the bacterial population next to the infected will express an antisense that if the lytic phages infect one of the alerted bacteria, it will be already prepared with the antisense. | The idea is that infected bacteria will send AHL to alarm their neighbors about the infection, afterwards the bacteria close to this place will express the asRNA, so if lytic phase of phages is faster than the transcription machinery and the toxin’s action time (This implies that the first infected bacteria will “lose” even when contains the construction), the bacterial population next to the infected will express an antisense that if the lytic phages infect one of the alerted bacteria, it will be already prepared with the antisense. | ||
+ | '''Sept 8th – 18th Design of the antisensense RNA''' | ||
+ | ---- | ||
- | |||
- | |||
We looked for any target that was mentioned as essential in the literature. We decided to attack phages in their replication process, because of the targets we found, so we reasoned that if the phage tries to replicate it’s genome the asRNA is going to block the process reducing dramatically the burst size or if the efficiency is high, there wont be any new phage. | We looked for any target that was mentioned as essential in the literature. We decided to attack phages in their replication process, because of the targets we found, so we reasoned that if the phage tries to replicate it’s genome the asRNA is going to block the process reducing dramatically the burst size or if the efficiency is high, there wont be any new phage. | ||
The target is NOT the RNA polymerase because we want it to express the kamikaze device. | The target is NOT the RNA polymerase because we want it to express the kamikaze device. | ||
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- | Concatenated: | + | Concatenated: <BR> |
CCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCC ATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTT | CCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCC ATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTT | ||
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At the end we send to synthesis the following sequence. | At the end we send to synthesis the following sequence. | ||
>Prefix_PLux_AsRNA(t3DNApol)_asRNA(t7SSBProt)_DoubleTerminator_Suffix | >Prefix_PLux_AsRNA(t3DNApol)_asRNA(t7SSBProt)_DoubleTerminator_Suffix | ||
- | + | GAATTCGCGGCCGCTTCTAGacctgtaggatcgtacaggttgacacaagaaaatggtttgttgatactcgaataaaCCTCA ATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCCATGTAAGCGTAAGGTT CAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTTccaggcatcaaata aaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacact ggctcaccttcgggtgggcctttctgcgtttataTACTAGTAGCGGCCGCTGCAG | |
DATE ?? | DATE ?? | ||
Mr.Gene shipment arrives. | Mr.Gene shipment arrives. | ||
+ | |||
+ | '''== New biobricks work ==''' | ||
Goals | Goals | ||
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-Join this product with all the other biobricks | -Join this product with all the other biobricks | ||
+ | '''September 21th.''' | ||
+ | ---- | ||
- | |||
High quality PCR performed to the synthesized biobricks. This bioparts just arived from GeneArt shipment | High quality PCR performed to the synthesized biobricks. This bioparts just arived from GeneArt shipment | ||
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PCR results were with the expected size for all three sequences. | PCR results were with the expected size for all three sequences. | ||
+ | '''Oct6th''' | ||
+ | ---- | ||
- | |||
- | |||
- | |||
- | |||
- | |||
PCR colony for those that are still white. We expected to find true transformants, but from 25 colonies, just one contains a PCR product of the expected size. | PCR colony for those that are still white. We expected to find true transformants, but from 25 colonies, just one contains a PCR product of the expected size. | ||
Almost all the few colonies with the pSB1T3 as a plasmid are red. In the case of those that are withe (the Kanamicine resistance conteiners) has just kanamicine ligated! we are surprised becouse we weren't expecting that the kanamicine resistance was able to clone without the colicin, just becouse of restriction enzymes combinations! | Almost all the few colonies with the pSB1T3 as a plasmid are red. In the case of those that are withe (the Kanamicine resistance conteiners) has just kanamicine ligated! we are surprised becouse we weren't expecting that the kanamicine resistance was able to clone without the colicin, just becouse of restriction enzymes combinations! | ||
- | Oct 7th | + | '''Oct 7th''' |
+ | ---- | ||
Plasmid extraction and digestion of the transformant colony whose PCR seemed to amplify a fragment of the expected size. | Plasmid extraction and digestion of the transformant colony whose PCR seemed to amplify a fragment of the expected size. | ||
Now we have a clone with the biobrick multipromoter_GFP inserted into the plasmid pSB1T3. | Now we have a clone with the biobrick multipromoter_GFP inserted into the plasmid pSB1T3. | ||
+ | '''Oct 8th''' | ||
+ | ---- | ||
- | |||
Preparation by dilution of Preffix and Suffix primers. | Preparation by dilution of Preffix and Suffix primers. | ||
High quality PCRs to E3 and E9, just to earn more of these DNA fragments in order to continue the efforts to clone them | High quality PCRs to E3 and E9, just to earn more of these DNA fragments in order to continue the efforts to clone them | ||
- | October 9th | + | '''October 9th''' |
+ | ---- | ||
+ | |||
Cut and dephosphatation of BBa_J04450 cut by EcoRI and PstI which expeled RFP from the plasmid. This new plasmid pSB4A5 is used due to the lack of transformants for the plasmid pSB4K5, both of them are low copy number plasmid. We reasoned that a high copy number plasmid may be the cause of our failure to clone E3 and E9 bacteriocines.<br> | Cut and dephosphatation of BBa_J04450 cut by EcoRI and PstI which expeled RFP from the plasmid. This new plasmid pSB4A5 is used due to the lack of transformants for the plasmid pSB4K5, both of them are low copy number plasmid. We reasoned that a high copy number plasmid may be the cause of our failure to clone E3 and E9 bacteriocines.<br> | ||
- | Oct 10th | + | '''Oct 10th''' |
+ | ---- | ||
+ | |||
Digestion with different enzyme combinations to different biobricks. | Digestion with different enzyme combinations to different biobricks. | ||
*E9 & E3 | *E9 & E3 |
Revision as of 00:58, 20 October 2009