Team:LCG-UNAM-Mexico/AbrahamJurnal
From 2009.igem.org
(Difference between revisions)
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Objectives: | Objectives: | ||
- | The final aim is to earn all the following devices and them all together into the plasmid we are suggesting, that | + | The final aim is to earn all the following devices and them all together into the plasmid we are suggesting, that will be extracted from P4 |
[[Image:Sistema.JPG]] | [[Image:Sistema.JPG]] | ||
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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> | 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> | ||
+ | '''June 27th-July 7th''' | ||
+ | ---- | ||
+ | Colony PCR from grown colonies and plasmid extraction from true possitives. | ||
+ | |||
+ | |||
+ | '''June 8th''' | ||
+ | ---- | ||
+ | My birthday! enjoying it was all my work. | ||
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'''July 14th''' | '''July 14th''' | ||
---- | ---- | ||
- | |||
Plan to ligate double terminators (BBa_B0015) at the end of the HSL making enzyme (BBa_C0261) the kanamycine resistance cassette (BBa_P1003) | 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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'''Aug 6th''' | '''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 | ||
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''' | '''Aug 7th''' | ||
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Ligations Number of colonies Lines | Ligations Number of colonies Lines | ||
BBa_R1062 + BBa_I13507 5 1 - 5 big | BBa_R1062 + BBa_I13507 5 1 - 5 big | ||
- | + | Cox-->BBa_J04450* 2 6, 7 big | |
BBa_C0261 + BBa_B0015 2 8, 9 big | BBa_C0261 + BBa_B0015 2 8, 9 big | ||
BBa_K116640 + BBa_K116640 8 11-18 big | BBa_K116640 + BBa_K116640 8 11-18 big | ||
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[[Image:Gels.jpg]] | [[Image:Gels.jpg]] | ||
- | + | *The arrow means that we want to clone the Cox gene into the plasmid pSB1T3 containing BBa_J04450 as a marker. | |
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. | ||
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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 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. | 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. | ||
- | + | ||
'''Sept 8th – 18th Design of the antisensense RNA''' | '''Sept 8th – 18th Design of the antisensense RNA''' | ||
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>Prefix_PLux_AsRNA(t3DNApol)_asRNA(t7SSBProt)_DoubleTerminator_Suffix | >Prefix_PLux_AsRNA(t3DNApol)_asRNA(t7SSBProt)_DoubleTerminator_Suffix | ||
GAATTCGCGGCCGCTTCTAGacctgtaggatcgtacaggttgacacaagaaaatggtttgttgatactcgaataaaCCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCCATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataTACTAGTAGCGGCCGCTGCAG | GAATTCGCGGCCGCTTCTAGacctgtaggatcgtacaggttgacacaagaaaatggtttgttgatactcgaataaaCCTCAATGTCACTTACGAGCATAATGCCCTCCTTTGGTTTCGTGCTAAATGATAATCATAAAGGCCACCCATGTAAGCGTAAGGTTCAGCGGTACCCAGCGCAGAGGTGAAAATCTTCTTAGCCATAATGTTAATCTCCTTTAGGTTTCGTTTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataTACTAGTAGCGGCCGCTGCAG | ||
+ | |||
'''September 17th''' | '''September 17th''' | ||
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Mr.Gene shipment arrives. In this point my task is going to be the work with the sequences that just have arrived. | 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 ==''' | ||
Goals | Goals | ||
Final goals: | Final goals: | ||
- | + | #Ensamble and prove the functionality of the kamikaze device. | |
- | + | #Get the time in wich a colicin, preferentially E3, kills E. coli c1-alpha. | |
- | + | #Clone the bioparts received from Gene Art into any iGEM plasmid vector. Send them to the Registry of standard biological parts. | |
Partial goals: | Partial goals: | ||
- | + | #Get the time in wich a colicin, preferentially E3, kills Escherichia coli c1-alpha | |
- | + | ##Clone both colicines into any iGEM vector. | |
- | + | ##Clone both colicines at the suffix of the biobrick BBa_R0010 in order to get an IPTG inducible device. | |
- | + | ##Measure the time of death after the induction with IPTG. | |
- | + | #Clone the MultiPromoter_GFP with all the death device | |
- | + | ##Insert the MP_GFP biobrick into any iGEM vector. | |
- | + | ##Insert the 2 colicines after the MP_GFP. | |
- | + | ##Join this product with all the other biobricks. | |
'''September 21th.''' | '''September 21th.''' | ||
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'''Oct 11th''' | '''Oct 11th''' | ||
---- | ---- | ||
- | I performed ligations according to the protocol of the following biobricks | + | I performed ligations according to the protocol of the following biobricks: |
- | ''' | + | E3 -->BBa_J04450, plasmid pSB4A5 |
+ | E9 -->BBa_J04450, plasmid pSB4A5 | ||
+ | E9 + BBa_P1003 -->BBa_J04450, plasmid pSB4A5 | ||
+ | E9 + BBa_P1003 -->BBa_J04450, plasmid pSB4A5 | ||
+ | BBa_R0010 + E3 (In the suffix of BBa_R0010) | ||
+ | BBa_R0010 + E9 (In the suffix of BBa_R0010) | ||
+ | |||
+ | he cultures containing BBa_P1003 are plated into Km antibiotic, thus it is supposed that if the ligation is good then we will se just true positives or new resistant colonies. | ||
+ | |||
+ | '''Octuber 13th-14th''' | ||
---- | ---- | ||
+ | The petri boxes now contain some colonies | ||
+ | E3 -->BBa_J04450* 3 colonies | ||
+ | E9 -->BBa_J04450* 0 colonies | ||
+ | E9 + BBa_P1003 -->BBa_J04450* many little colonies | ||
+ | E9 + BBa_P1003 -->BBa_J04450* many little colonies | ||
+ | BBa_R0010 + E3 2 colonies | ||
+ | BBa_R0010 + E9 6 colonies | ||
+ | Negative control 0 colonies | ||
+ | Ligation without insert control 1 colony | ||
+ | |||
+ | *Low copy number plasmid pSB1A2 | ||
+ | I managed to do colony PCR and placed the boxes with BBa_P1003 into the incubator again, this is because we suspect that the toxines are being detrimental for E. coli's fitness and that is the reason to observe that their growth is so slow. | ||
+ | |||
+ | [[Image:UltimoIntento.jpg]] | ||
+ | |||
+ | Lane 1.- DNA ladder | ||
+ | Lane 2.- Colony PCR positve control. | ||
+ | Lanes 3-4.- BBa_R0010 + E3 | ||
+ | Lanes 5-6.- E3 -->BBa_J04450 | ||
+ | Lane 7.- PCR Negative control | ||
+ | Lane 8.- PCR of E3 from Mr Gene shipment. | ||
+ | Lanes 9-15.- BBa_R0010 + E9 | ||
+ | |||
+ | We can see in lines 3, 4, 9-12 with a band arround 200 bp, these are from the BBa_R0010 promoter, so again, we just obtained clones with one of the two inserts even when the restriction enzymes used are supposed to avoid this result. | ||
+ | |||
+ | |||
+ | '''Octuber 15th-16th''' | ||
+ | ---- | ||
+ | We placed the biggest colonies of the plates with Km into the antibiotic for wich they have resistance into the plasmid, Ampiciline. In parallel we performed colony PCR. | ||
+ | |||
+ | [[Image:LastPCRs.jpg]] | ||
+ | |||
+ | The results were negatives :( and the cultures in Amp were succesfull. But the morphology of the colonies don't look like E. coli. | ||
+ | At the same time we observe the original plates with some fungi, so we conclude that the little colonies were contaminated with some mushroom. | ||
+ | |||
+ | '''Octuber 18th''' | ||
+ | ---- | ||
+ | Due to the failed efforts to clone the colicines, we will try again after the wiki fresh. Maybe with the topo cloning technique. | ||
+ | |||
+ | '''September 19th-20th''' | ||
+ | ---- | ||
+ | Registry of the bioparts into the Registry of Standard Biological parts and in work in the wiki. :) | ||
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Revision as of 17:19, 21 October 2009