Team:LCG-UNAM-Mexico/AbrahamJurnal
From 2009.igem.org
(Difference between revisions)
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Colony PCR<br> | Colony PCR<br> | ||
- | Take the colony and resuspend in 200uL of TE 10:1 NaCl 10M | + | Take the colony and resuspend in 200uL of TE 10:1 NaCl 10M<br> |
- | Heat the suspension for 10 minutes at 95°C | + | Heat the suspension for 10 minutes at 95°C<br> |
- | Centrifugue for 2 minutes at 14000 RPM | + | Centrifugue for 2 minutes at 14000 RPM<br> |
- | Use 10uL of this DNA extract as template for the PCR | + | Use 10uL of this DNA extract as template for the PCR<br> |
The mixture to perform the PCR is:<br> | The mixture to perform the PCR is:<br> | ||
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[[Image:Sistema.JPG]] | [[Image:Sistema.JPG]] | ||
- | I learned to obtain the plasmid send by iGEM in the 2009 kit plates, and to transform it | + | I learned to obtain the plasmid send by iGEM in the 2009 kit plates, and to transform it, the biobricks we are trying to obtain are: |
+ | |||
+ | BBa_R1062 Promoter activated by LuxR-HSL complex <Br> BBa_I1352 RFP constitutively expressed and repressed with tetracycline<br> BBa_J37033 LuxR protein<br> BBa_C0261 AHL making enzyme<br> BBa_B0015 Double transcriptional terminator<br> BBa_P1003 Kanamycine ressistance casette<br> BBa_J04450 mRFP<br> | ||
+ | |||
+ | |||
'''July 10th''' | '''July 10th''' | ||
---- | ---- | ||
- | We transformed the plasmids coming in the 2009 kit plates | + | We noticed that we needed some other biobricks in order to test the function of our principal components, so we transformed the plasmids coming in the 2009 kit plates:<br> |
- | BBa_I715038 ready to use T7 RNA polymerase inducible with IPTG | + | |
- | BBa_K093005 RBS-RFP | + | BBa_I715038 ready to use T7 RNA polymerase inducible with IPTG |
- | BBa_I13507 RBS-RFP-Transcriptional terminator | + | BBa_K093005 RBS-RFP |
+ | BBa_I13507 RBS-RFP-Transcriptional terminator | ||
The T7 RNA polymerase is going to be used to test the functionality of the final device and of the partial constructions with this promoter. | The T7 RNA polymerase is going to be used to test the functionality of the final device and of the partial constructions with this promoter. | ||
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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- | + | 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-HSL dimer (BBa_R1062). |
'''July 14th''' | '''July 14th''' | ||
---- | ---- | ||
- | Plan to ligate double terminators (BBa_B0015) at the end of the | + | Plan to ligate double terminators (BBa_B0015) at the end of the HSL making enzyme (BBa_C0261) the kanamycine resistance cassette (BBa_P1003) |
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---- | ---- | ||
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 | + | The idea is that infected bacteria will send HSL 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. |
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We met Dr. José Luis Reyes Taboada who is expert in the field of RNAs. With his guidance we looked for structures in RNAFold with high Gibbs energy values (the less negative, the better), we also checked that the structures were not blocking the RBS. | We met Dr. José Luis Reyes Taboada who is expert in the field of RNAs. With his guidance we looked for structures in RNAFold with high Gibbs energy values (the less negative, the better), we also checked that the structures were not blocking the RBS. | ||
+ | |||
+ | We planned to include a marker such as mRFP in the 5' side of the antisense, but we though that the translation of this protein would interfer with the function of the antisense, thus we will include this biobrick induced by the complex LuxR-HSL (as well as the mRFP), but in different cistrons. | ||
Why not to use the same target mRNA for both phages? Because the upstream sequences of the essential genes are not conserved more than 90%, and the efficiency of silencing would decrease dramatically for one if we take the antisense sequence of the other. | Why not to use the same target mRNA for both phages? Because the upstream sequences of the essential genes are not conserved more than 90%, and the efficiency of silencing would decrease dramatically for one if we take the antisense sequence of the other. | ||
- | Why not use the two different sequences but for the same target in the two phages (for example, both DNA polymerases)? Because they are more similar than any other pair of sequences, so they would interfere with each other. | + | Why not use the two different sequences but for the same target in the two phages (for example, both DNA polymerases)? Because they are more similar than any other pair of sequences, so they would interfere with each other. The alternative strategy is atack the same process but with different components |
- | The final | + | The final choices are the following sequences: |
GGGTGGCCTTTATGATTATCATTTAGCACGAAACCAAAGGAGGGCATTATGCTCGTAAGTGACATTGAGG | GGGTGGCCTTTATGATTATCATTTAGCACGAAACCAAAGGAGGGCATTATGCTCGTAAGTGACATTGAGG | ||
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Their reverse complement sequences are: | Their reverse complement sequences are: | ||
- | |||
*CCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCC. | *CCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCC. | ||
Form two possible secondary structures of -10 and -11 kcal/mol. | Form two possible secondary structures of -10 and -11 kcal/mol. | ||
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GAATTCGCGGCCGCTTCTAGacctgtaggatcgtacaggttgacacaagaaaatggtttgttgatactcgaataaaCCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCCATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataTACTAGTAGCGGCCGCTGCAG | GAATTCGCGGCCGCTTCTAGacctgtaggatcgtacaggttgacacaagaaaatggtttgttgatactcgaataaaCCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCCATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataTACTAGTAGCGGCCGCTGCAG | ||
- | + | '''September 17th''' | |
- | Mr.Gene shipment arrives. | + | ---- |
+ | Mr.Gene shipment arrives. In this point my task is going to be the work with the sequences that just have arrived. | ||
'''== New biobricks work ==''' | '''== New biobricks work ==''' | ||
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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 | ||
- | MultiPromoter T3/T7_GFP (furthermore refered as MP_GFP)-- We recieved 5ng of plasmid containing this biobrick | + | MultiPromoter T3/T7_GFP (furthermore refered as MP_GFP)-- We recieved 5ng of plasmid containing this biobrick<br> |
- | E3 RNase domain (furthermore called E3)-- We recieved just 0.1ng of PCR product of this biobrick | + | E3 RNase domain (furthermore called E3. Later registered as BBa_K242000)-- We recieved just 0.1ng of PCR product of this biobrick<br> |
- | E9 DNase domain (furthermore called E9)-- We recieved just 0.1ng of PCR product of this biobrick | + | E9 DNase domain (furthermore called E9. Later registered as BBa_K242029)-- We recieved just 0.1ng of PCR product of this biobrick<br> |
(Check Aug 6th for details) | (Check Aug 6th for details) | ||
PCR results were with the expected size for all three sequences. | PCR results were with the expected size for all three sequences. | ||
- | + | ||
- | ''' | + | [[Image:geneart.jpg]] |
+ | |||
+ | Line 1.- DNA ladder | ||
+ | Line 2.- E9 DNase domain | ||
+ | Line 3.- E3 RNase domain | ||
+ | Line 4.- MP_GFP (there's a band in the size of the plasmid length) | ||
+ | |||
+ | '''September 21th.''' | ||
---- | ---- | ||
+ | |||
+ | '''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! | ||
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Double transcriptional terminator will be inserted just before the colicines, as a control to stop basal transcription. | Double transcriptional terminator will be inserted just before the colicines, as a control to stop basal transcription. | ||
+ | |||
+ | We also started the registration of the biobricks we will provide to the Registry of Standard Biological Parts | ||
+ | |||
+ | |||
+ | '''Oct 11th''' | ||
+ | ---- | ||
+ | I performed ligations according to the protocol of the following biobricks | ||
+ | |||
+ | '''Oct 19th''' | ||
+ | ---- | ||
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Revision as of 15:27, 20 October 2009