Difference between revisions of "Team:JiangnanU China/Parts"

250bp constitutive promoter
RsmH can detect the intensity of the fluorescence of gfp.

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 weakest, and whose RBS strength is 0.35% of BBa_B0034. By analyzing their sequence, 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.

250bp inducible promoter
PputA is an inducible promoter. When E.coli is infected by a phage, both report and respond 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.

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 fluorescence 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.

708bp report gene
The mCherry can express red fluorescence protein and it is used as a report gene in the process of reporting the number of cells.

996bp
The Gluconate repressor gntR, is a transcription factor that negatively regulates the operon involved in the catabolism of d-gluconate via the Entner-Doudoroff pathway and represses genes involved in the different systems related to d-gluconate uptake: gluconate I and gluconate II.

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.

462bp
Overexpression of rzpD causes an abnormal biofilm architecture.

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.

501bp
NuoE is part of the soluble fragment of NADH dehydrogenase I, which represents the electron input part of the enzyme.

48bp terminator
T7 terminator is used to quantify the level of expression in E. coli.

717bp reporter
The gfp can express green fluorescent protein, which can be used as a report gene in the process to report the number of cells.

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 outbreak 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, both report and respond gene circuits are induced to express at the same time. When a phage infects E. coli into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to express the anti-proteins and the green fluorescent protein gene gfp. If the anti-protein successfully resists phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the anti-protein fails to kill phages, it means that phages will continue to infect bacteria. And 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 and the downstream toxic protein gene so that the bacteria will display red fluoresce and lyse cells before the assembly of phages.

5097bp

The respond gene circuit is composed of an inducible promoter PputA and anti-phage protein genes of the latent 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, both report and respond gene circuits are induced to express at the same time. When a phage infects E. coli into the latent period (about 5 min), the inducible promoter PputA will induce the bacteria to express the resistant protein abpAB and gntR, and the 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 anti-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 and downstream toxic protein gene protegrin-1 so that the bacteria will display red fluoresce and lyse cells before the assembly of phages.