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Our Field of Dreams
We will produce a BioBrick part for the Cytochrome P450 Flavonoid-3`,5`-hydroxylase (F-3`-5`-H) gene. Flavonoid-3`,5`-hydroxylase is the key enzyme in the synthesis of 3`-5`-hydroxylated anthocyanins that is required to produce the blue pigmentation in Phalaenopsis (orchid) flowers. F-3`-5`-H BioBrick part will be expressed in Escherichia coli, so that it will produce these anthocyanins when the colonies are exposed to chemicals in the biosynthesis pathway for the production of anthocyanins.
To produce the F-3`-5`-H BioBrick part, we will extract DNA from Phalaenopsis leaf cuttings and RNA from the flower petals. The cuttings will be obtained from the University of Northern Iowa Greenhouse and Botanical Center. This orchid has our target gene which is evidenced by the purple blooms on the plant. We will be extracting DNA from the leaf cutting with the Hop DNA Isolation  protocol. The extracted DNA will be amplified and with primers designed from the mRNA sequence on GenBank (accession number DQ 148458) . The primers will also have additions on them that will add the BioBrick prefix and suffix. The amplified DNA will be sequenced by University of Iowa and analyzed to identify if there are any introns or any restriction sites for Spe1, Xbal, EcoR1 and Pst1. The restriction sites and introns need to be removed to make this gene BioBrick compatible. If any introns are identified, they will be removed by site-directed mutagenesis. If there are restriction sites that need to be removed, the sequence will be reanalyzed to see if removing the site or changing the site to another codon will maintain the gene‘s ability to transcribe a functional protein.
RNA will be extracted with Trizol product protocol from the manufacturer. The RNA will be amplified by reverse PCR with primers designed from the sequence from GenBank and that will also add on the BioBrick prefix and suffix.
The new BioBrick genes from RNA and DNA will be tested by inserting it into E. coli. Two different vectors will be used, PSB1A3 and PSB1A7. The plasmids will be isolated from E. coli using Miniprep/GET buffer protocol  and cut with Xbal and SpecI as will the F3`-5`-H BioBrick part. This will ensure that there are “sticky ends” so that the target gene can be added to the cut plasmid. We will be using E. coli promoter Bba_R0040 TetR repressible promoter which is normally on, and is only shut off in the presence of tetracycline and its derivatives. Once the promoter, target gene, and plasmid are assembled, they will be reinserted into chemically competent E. coli cells. The transformed E. coli will be screened for successful insertion, by growing them on media with Kanamycin. If the colonies survive, the transformation was successful. The effectively transformed colonies will be grown on media containing naringenin, which is a precursor in the anthocyanins biosynthesis pathway as illustrated in figure 1.1 below. If this new BioBrick works, the colonies should turn blue.
Figure 1.1 Blue Anthocyanin Pathway
The goal of this project was to produce a BioBrick compatible part from the cytochrome P450 flavonoid-3`-5`-hydroxylase gene extracted from Phalaenopsis plant to be functionally expressed in Escherichia coli. We were going to obtain this part by extracting DNA from the leaves and RNA from the flowers. Next we would amplify the gene with primers (F1BBa and R1BBa) that were designed using the GENBANK sequence (accession number DQ148458) for mRNA of the target gene and the required BioBrick prefix and suffix. The new BioBrick part would be ligated into plasmids pSB1A3 and pSB1A7 extracted from the IGEM 2007 Parts Registry. The vectors would be electroporated into E. coli colonies and the successfully transformed colonies would be grown on naringenin enhanced medium to produce the blue flavonoid.
After DNA extraction using Hops DNA Isolation protocol from Dr. Schwekendiek’s lab the DNA was tested without its primers to prove that DNA was obtained (see figure 1). Then target gene was amplified using designed primers(F1BBa and R1BBa) and a band was observed between 2000 and 3000 base pairs. The gene fragment was longer than was expected for the target gene, which should have been around 1500 base pairs. The fragment was sequenced by Iowa State University and was analyzed using NCBI’s BLAST to confirm that the target gene was amplified. The forward read of 642 bases had a 100% match to the target gene and the reverse read was a 92% match for 240 bases. However a complete read was not obtained due to the length of the gene fragment since sequencing becomes less reliable after 1000 bases. Since the gene was longer than was expected from the mRNA sequence this indicated that there was at least one intron present in the DNA sequence.
RNA was extracted using RNeasy Extraction Kit from QIAGEN and amplified by reverse transcription PCR. A band was observed between 1500 and 2000 base pairs (see figure 2) which was the expected length of the cDNA fragment. Since the DNA amplification had a match for the target gene and the same primers (F1BBa and R1BBa) were used, the cDNA was not sequenced.
The plasmids pSB1A3 and pSB1A7 were extracted from glycerol stocks of a strain of E. coli, DH5alpha, available in Dr. Schwekendiek’s lab using MiniPrep/GET Buffer Protocol from OpenWetWare.org. The plasmid pSB1A3 should show a band at approximately 2100 base pairs, and pSB1A7 should show a band around 2400 base pairs. Instead bands were seen around 600 base pairs for both plasmids. The same results were seen after being repeated. Since the same results were obtained with these samples, pSB1A3 and pSB1A7 were extracted from the 2007 IGEM Parts Registry. The parts from the registry turned out not to be compatible with the DH5alpha strain because the plasmids contain a death gene which makes them toxic to the DH5alpha cells. Then glycerol stocks, from MIT, of a different strain of E. coli, DB3.1, containing plasmids pSB1A3 and pSB1A7, were used. Again bands of the incorrect size were observed. The enzyme that was being used could potentially be the problem, so fast digest enzymes were substituted and a new extraction kit GeneJet MiniPrep from QIAGEN was used. A band around 2000 base pairs for pSB1A7 and a band around 2500 base pairs for pSB1A3 were observed and this was the reverse of what it should have been. This suggested that the samples could have been mislabeled at some point in previous attempts. This was verified by PCR using VF2 and VR primers to amplify a segment of DNA present in both plasmids, but differing in length; for pSB1A3 this segment of DNA is 316 base pairs long and in pSB1A7 it is 590 base pairs long. A band around 1000 base pairs for pSB1A3 and a band around 350 base pairs for pSB1A7 were present. Concluded from these results was that pSB1A7 was not the target plasmid and pSB1A3 could be the target plasmid if the samples had been mixed up. To verify if the plasmid samples were in fact mixed up, the extracted plasmid DNA was digested using SpeI and XbaI. This indicated that the MIT samples of pSB1A7 were not in fact the pSB1A7 plasmid, as described in the parts registry, but instead was an unknown plasmid. Also the MIT samples of pSB1A3 did contain the pSB1A3 plasmid as described in the registry.
Due to the problems with extracting pSB1A7 and pSB1A3, a plasmid that contained a promoter was decided to be used instead. The plasmid pSB1A2 with promoter BBa_R0040, BBa_R0051, BBa_R001 was extracted from IGEM 2007 and Spring 2008 Parts Registry using the protocol suggested by the Parts Registry. The plasmid was double digested with Pst1 and Spe1 to open for ligation with the target gene.
The PCR amplified gene from DNA was digested with Xbal and Pst1 and the double digested sample showed two bands instead of the expected one. This could be due to the presence of an intron which has a restriction site. The two fragments were ligated into plasmid pSB1A2 and electroporated into DH5alpha strain of E. coli. Random colonies from the transformation were cultured in LB to check for relegation of the whole target gene. When the plasmids were extracted from the LB cultures, there were no results seen due to the length of time that the samples were in LB.
The cDNA was double digested with Xbal and Pst1 and the same results as in the DNA PCR product. Two bands in addition to the undigested band were seen indicating that were was a missed restriction site in the sequence.
Due to time constraints, this project is currently on hold. For future work, the sequence on GENBANK will need to be reanalyzed using more than one reference to locate any restriction sites. The full sequence of the DNA PCR product would need to be obtained. This could be done by designing more primers from the forward and reverse reads to acquire the rest of the sequence.
The Journal is posted in the calendar section at the top of this page by the team members of Emblazon.