Difference between revisions of "Team:JiangnanU China/Parts"
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| − | + | One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators. These proteins contain a DNA-binding HTH domain at the N-terminus of the protein and an effector-binding and/or oligomerisation domain at the C-terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription[1]. | |
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recombinant plasmid that connected <i>abpA</i> and <i>abpB</i> at the same time. Then we used IPTG to | recombinant plasmid that connected <i>abpA</i> and <i>abpB</i> at the same time. Then we used IPTG to | ||
induce the | induce the | ||
| − | expression of anti-protein and verified the resistant function of the protein. | + | expression of anti-protein and verified the resistant function of the protein.[2] |
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<img src="https://static.igem.org/mediawiki/2019/8/81/T--JiangnanU_China--parts_2000.png" | <img src="https://static.igem.org/mediawiki/2019/8/81/T--JiangnanU_China--parts_2000.png" | ||
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| + | References[1-2] | ||
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| + | [1] Hoskisson P A , Sébastien Rigali. Chapter 1: Variation in form and function the helix-turn-helix regulators of the GntR superfamily[J]. Advances in applied microbiology, 2009, 69(69):1-22. | ||
| + | <br /> | ||
| + | [2] Yasui R, Washizaki A, Furihata Y, Yonesaki T, Otsuka Y: AbpA and AbpB provide anti-phage activity in <i>Escherichia coli</i>. Genes Genet Syst 2014, 89(2):51-60. | ||
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Latest revision as of 02:26, 22 October 2019
Parts
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Basic Parts
BBa_K3137000 : PrsmH
250bp constitutive promoter
We ligated promoter rsmH with gfp to measure the fluorescence intensity of GFP.
We ligated promoter rsmH with gfp to measure the fluorescence intensity of GFP.
BBa_K3137001 : Improved RBS 4
11bp RBS
As we all know, RBS refers to a sputum-rich untranslated region upstream of the initiation codon AUG. BBa_B0033 is a weaker RBS based on Ron Weiss thesis. Compared with BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0034, BBa_B0033 is the weakest, and whose RBS strength is 0.35% of BBa_B0034. By analyzing their sequences, we found that BBa_B0033 has fewer purines and no not have AAA sequence. SO we designed the RBS by adjusting the proportion of bases.
Then we ligated the two RBS with promotor rsmH (BBa_K3137000) and gene gfp (BBa_K3137011) and transform into E. coli BL21 to compare their green fluorescence. And take E. coli BL21 as a control. The figure below shows that BBa_K3137001 has the stronger fluorescently protein expression than BBa_B0033, that means it’s efficient to increase the protein expression by increasing the number of purines in the RBS sequence, which is beneficial to the binding of ribosomes.
As we all know, RBS refers to a sputum-rich untranslated region upstream of the initiation codon AUG. BBa_B0033 is a weaker RBS based on Ron Weiss thesis. Compared with BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0034, BBa_B0033 is the weakest, and whose RBS strength is 0.35% of BBa_B0034. By analyzing their sequences, we found that BBa_B0033 has fewer purines and no not have AAA sequence. SO we designed the RBS by adjusting the proportion of bases.
Then we ligated the two RBS with promotor rsmH (BBa_K3137000) and gene gfp (BBa_K3137011) and transform into E. coli BL21 to compare their green fluorescence. And take E. coli BL21 as a control. The figure below shows that BBa_K3137001 has the stronger fluorescently protein expression than BBa_B0033, that means it’s efficient to increase the protein expression by increasing the number of purines in the RBS sequence, which is beneficial to the binding of ribosomes.
BBa_K3137002 : PputA
250bp inducible promoter
PputA is an inducible promoter. When E.coli is infected by a phage, both report and response gene circuits are induced to express at the same time. When a phage infects Escherichia coli into the latent period (about 5 min), PputA will induce the bacteria to express anti-protein and display green fluorescence.
PputA is an inducible promoter. When E.coli is infected by a phage, both report and response gene circuits are induced to express at the same time. When a phage infects Escherichia coli into the latent period (about 5 min), PputA will induce the bacteria to express anti-protein and display green fluorescence.
BBa_K3137003 : PglcF
250bp inducible promoter
PglcF is an inducible promoter that works when phages infect bacterial for around 20 min. If the anti-proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the anti-proteins cannot kill phages, the phages will continue to infect. When phages infect bacteria into the burst period (about 20 min), the inducible promoter PglcF will induce expression of the red fluorescent protein gene mCherry and the downstream toxic protein gene protegrin-1, which will make the bacteria display red fluorescence and lyse cells before the assembly of phages.
PglcF is an inducible promoter that works when phages infect bacterial for around 20 min. If the anti-proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the anti-proteins cannot kill phages, the phages will continue to infect. When phages infect bacteria into the burst period (about 20 min), the inducible promoter PglcF will induce expression of the red fluorescent protein gene mCherry and the downstream toxic protein gene protegrin-1, which will make the bacteria display red fluorescence and lyse cells before the assembly of phages.
BBa_K3137004 : mCherry
708bp reporter gene
The mCherry can express red fluorescent protein and it is used as a reporter gene in the process of reporting the number of cells.
The mCherry can express red fluorescent protein and it is used as a reporter gene in the process of reporting the number of cells.
BBa_K3137005 : gntR
996bp
One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators. These proteins contain a DNA-binding HTH domain at the N-terminus of the protein and an effector-binding and/or oligomerisation domain at the C-terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription[1].
One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators. These proteins contain a DNA-binding HTH domain at the N-terminus of the protein and an effector-binding and/or oligomerisation domain at the C-terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription[1].
BBa_K3137006 : abpAB
3803bp anti-protein
Gene abpAB is from E.coli genome which express anti-proteins that are resistant to T2,T4,T7 and λ phages. We obtained abpA and abpB by PCR from the genome of E.coli BL21 and constructed recombinant plasmid that connected abpA and abpB at the same time. Then we used IPTG to induce the expression of anti-protein and verified the resistant function of the protein.[2]
Gene abpAB is from E.coli genome which express anti-proteins that are resistant to T2,T4,T7 and λ phages. We obtained abpA and abpB by PCR from the genome of E.coli BL21 and constructed recombinant plasmid that connected abpA and abpB at the same time. Then we used IPTG to induce the expression of anti-protein and verified the resistant function of the protein.[2]
BBa_K3137007 : rzpD
462bp
Overexpression of rzpD causes an abnormal biofilm architecture.
Overexpression of rzpD causes an abnormal biofilm architecture.
BBa_K3137008 : yhjH
yhjH 768bp
YhjH protein contains a EAL domain to catalyze c-di-GMP into GMP, so as to regulate the levels of c-di-GMP.
YhjH protein contains a EAL domain to catalyze c-di-GMP into GMP, so as to regulate the levels of c-di-GMP.
BBa_K3137009 : nuoE
501bp
NuoE is part of the soluble fragment of NADH dehydrogenase I, which represents the electron input part of the enzyme.
NuoE is part of the soluble fragment of NADH dehydrogenase I, which represents the electron input part of the enzyme.
BBa_K3137010 : T7 terminator
48bp terminator
T7 terminator is used to quantify the level of expression in E. coli.
T7 terminator is used to quantify the level of expression in E. coli.
BBa_K3137011 : gfp
717bp reporter gene
The gfp can express green fluorescent protein, which can be used as a reporter gene in the process of reporting the number of cells.
The gfp can express green fluorescent protein, which can be used as a reporter gene in the process of reporting the number of cells.
Composite Part
BBa_K3137013 : report circuit
1666bp
The report gene circuit consists of two periods of phage infection. In incubation period (about 5 min), there is an inducible promoter PputA and a green fluorescent protein gene gfp. In burst period (about 20 min), there is an inducible promoter PglcF, and a red fluorescent protein gene mCherry.
When E. coli is infected by a phage, report gene circuit is induced to express. When a phage infects E. coli into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to expressthe green fluorescent protein gene gfp.
If the resistant proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the resistant proteins fail to kill phages, it means that phages will continue to infect bacteria, when it comes to the burst period (around 20 min) of phage infection, the inducible promoter PglcF will activate the red fluorescent protein gene mCherry. so that the bacteria will display red fluoresce and lyse cells before the assembly of phages.
BBa_K3137014 : response circuit
5097bp
The responce gene circuit is composed of an inducible promoter PputA and anti-phage protein genes of the incubation period (about 5 min), and an inducible promoter PglcF and a toxic protein gene protegrin-1 of the burst period (about 20 min).
When E. coli is infected by a phage, responce gene circuit is induced to express. When a phage infects E. coli into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to express the resistant proteins.
If the resistant proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the resistant proteins fail to kill phages, it means that phages will continue to infect bacteria, when it comes to the burst period (around 20 min) of phage infection, the inducible promoter PglcF will activate the expression of downstream toxic protein gene so that the bacteria will lyse cells before the assembly of phages.
References[1-2]
[1] Hoskisson P A , Sébastien Rigali. Chapter 1: Variation in form and function the helix-turn-helix regulators of the GntR superfamily[J]. Advances in applied microbiology, 2009, 69(69):1-22.
[2] Yasui R, Washizaki A, Furihata Y, Yonesaki T, Otsuka Y: AbpA and AbpB provide anti-phage activity in Escherichia coli. Genes Genet Syst 2014, 89(2):51-60.
[1] Hoskisson P A , Sébastien Rigali. Chapter 1: Variation in form and function the helix-turn-helix regulators of the GntR superfamily[J]. Advances in applied microbiology, 2009, 69(69):1-22.
[2] Yasui R, Washizaki A, Furihata Y, Yonesaki T, Otsuka Y: AbpA and AbpB provide anti-phage activity in Escherichia coli. Genes Genet Syst 2014, 89(2):51-60.
