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Team:UST Beijing/Footsteps
From 2014.igem.org
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| + | var arrMessages = ["Red fluorescent protein, which is also called RFP, is widely used as indicator or reporter gene in iGEM and other researches because it is easy for observation and measurement. During our experiments, RFP was also used in our experiment design. Parts from igem delivered plates, BBa_J04450, pSB1AC3 (http://parts.igem.org/Part:BBa_J04450), is an expression vector with highly engineered RFP gene sequence. But in our experiment, we found some very interesting character of this part. After that, we repeated the same procedures to make eliminate possible operation errors which make us believe that the phenomenon we observed can be reproducible. Since temperature was the only factor that has influenced the expression rate of rfp, there are several possible mechanisms our team members have proposed to explain it. The first kind of opinion is that, under lower temperature, the cells’ metabolism rate is also lowered, which means slower rate of transcription and expression rate. When cells facing with such problems, they might choose to appropriate their limited energy and material to the expression of house-keeping genes. On the other hand, 28 centigrade temperature is probably not cold enough to cause too much survival pressures for bacteria but may induce some other change to the RFP gene transcription and expression. Long hairpin structure caused by long reverted repeat sequences might hinder the movement of ribosome along mRNA, which means the lower rate of expression. ", | ||
| + | "In this paper published in 2006, researchers believe that the secondary structure near the start codon AUG of mRNA is essential form the expressing rate of human genes in Ecoli. Long hairpin structures near the start codon could form quickly after the initiation of transcription and become obstacles to ribosome movement along mRNA and then the elongation of peptide. This article make us believe that we should analyze the inverted repeats sequence especially the ones in the neighborhood of start codon. Then we used the software RNA structure and an online tool(http://rna.tbi.univie.ac.at) to predict the possible secondary structures in the RFP gene sequence. ", | ||
| + | "Results from both online or software secondary structure prediction show that A lot of secondary structures exist in the gene sequence. Since RFP has been used in a wide range not only in igem competition, its expression rate instability could be a problem for its application. In previous igem competitions, RFP have been used as reporter gene of Arsenic reporter, which could the host bacteria as Arsenic concentration sensitive color indicator. If the expression rate is significantly interfered by temperature, its indicator utility could be limited. In order to eliminate the possible hairpin structures, new gene sequence should be designed. ", | ||
| + | "In this article published in 2001, melting temperature of short paired DNA sequences, primers and target DNA sequences in PCR for example, can be influenced by many factors including the length of paired sequences, GC content and arrangement and cation concentration in the solution. The authors have also developed a series of mathematic formula to estimate the Tm of double DNA stands, which could be especially useful for primers design in PCR. What’s more, the article also gave us a hint that the addition of Dimethyl Sulfoxide can decrease the Tm of the double strand sequence. Such strategy inspire us to design a new experiment to confirm our hypothesis that the lower expression rate of RFP was caused by secondary structures of the mRNA.", | ||
| + | "Ecoli strain with psb1ac3-RFP plasmid was cultured in 2ml LB. There are two groups under different temperature 27centigrade(upper) and 37 centigrade(lower), respectively. The content of DMSO is 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, and 20%(just for second group), respectively. The results indicate that DMSO concentration at the 5-6% percent can successfully increase the expression rate of RFP. On the other hand, higher concentration of DMSO show toxicity to cells.", | ||
| + | "20℃ 72h 200rpm, DMSO as labeled.", | ||
| + | "30℃ 72h 200rpm, DMSO as labeled."] | ||
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| + | <p align="left" id="sticksCarouselMessage">Red fluorescent protein, which is also called RFP, is widely used as indicator or reporter gene in iGEM and other researches because it is easy for observation and measurement. During our experiments, RFP was also used in our experiment design. Parts from igem delivered plates, BBa_J04450, pSB1AC3 (http://parts.igem.org/Part:BBa_J04450), is an expression vector with highly engineered RFP gene sequence. But in our experiment, we found some very interesting character of this part. After that, we repeated the same procedures to make eliminate possible operation errors which make us believe that the phenomenon we observed can be reproducible. Since temperature was the only factor that has influenced the expression rate of rfp, there are several possible mechanisms our team members have proposed to explain it. The first kind of opinion is that, under lower temperature, the cells’ metabolism rate is also lowered, which means slower rate of transcription and expression rate. When cells facing with such problems, they might choose to appropriate their limited energy and material to the expression of house-keeping genes. On the other hand, 28 centigrade temperature is probably not cold enough to cause too much survival pressures for bacteria but may induce some other change to the RFP gene transcription and expression. Long hairpin structure caused by long reverted repeat sequences might hinder the movement of ribosome along mRNA, which means the lower rate of expression. </p> | ||
Latest revision as of 16:00, 16 October 2014
RFP Parts Evaluation
Evaluation and characterization of a highly engineered RFP gene (BBa_J04450): thermo sensitive expression pattern and secondary structure of mRNA.
Red fluorescent protein, which is also called RFP, is widely used as indicator or reporter gene in iGEM and other researches because it is easy for observation and measurement. During our experiments, RFP was also used in our experiment design. Parts from igem delivered plates, BBa_J04450, pSB1AC3 (http://parts.igem.org/Part:BBa_J04450), is an expression vector with highly engineered RFP gene sequence. But in our experiment, we found some very interesting character of this part. After that, we repeated the same procedures to make eliminate possible operation errors which make us believe that the phenomenon we observed can be reproducible. Since temperature was the only factor that has influenced the expression rate of rfp, there are several possible mechanisms our team members have proposed to explain it. The first kind of opinion is that, under lower temperature, the cells’ metabolism rate is also lowered, which means slower rate of transcription and expression rate. When cells facing with such problems, they might choose to appropriate their limited energy and material to the expression of house-keeping genes. On the other hand, 28 centigrade temperature is probably not cold enough to cause too much survival pressures for bacteria but may induce some other change to the RFP gene transcription and expression. Long hairpin structure caused by long reverted repeat sequences might hinder the movement of ribosome along mRNA, which means the lower rate of expression.
