Team:CDHSU-CHINA/Experiments

The following is our experimental procedure:
1.The isolation of RNA of Miracle fruit: First, we are going collect the RNA of Miracle fruits we bought from online shop. The RNA of miracle fruits could only be expressed in the mature miracle fruit. 2.Then we transcribe the RNA to cDNA
3.Design the PCR primer according the sequence of miracle fruit’s DNA on NCBI 4.Use PCR method to amplify the miracle fruit’s DNA 5.Do the DNA sequencing and compare the result to the standard DNA sequence
6.Digest the the amplified Miracle fruit’s DNA, connect the digested part to T-carrier. Then transfer the re-constructed plasmid to competent cell of E.coli 7.Smear the E. coli with re-constructed plasmid on the plate with Ampicillin, select the E. coli that could grow on the plate normally. Then we do colony PCR of the selected E. coli. After the PCR operation, the DNA of miracle fruits that shows amplification is confirmed to be positive clone. Compare the sequence of miracle fruit’s DNA gained through amplification to standard one to check if there is a mutation 8.Examine the pve5523(the expressing vector of lactobacillus) to make sure it is usable
9.Collect the plasmid of pve5523 and E. coli with re-constructed plasmid, do the double-digestion, recycle the fragments of MF’s DNA located in the E.coli plasmid with re-constructed plasmid and fragments of pve5233 after the digestion individually 10.Use T4 ligase to connect fragments of MF’s DNA to the fragments of pve5233(both of fragments gained from recycle), then we get a new plasmid involves those two fragments
11.Transfer the new re-constructed plasmid into the competent cell of E.coli, smear the new E.coli above on the plate with clarithromycin. Then we could select the E.coli that could normally grow, and do the colony PCR of the normal growth E.coli. After the colony PCR, the E.coli has amplified DNA of MF is comfirmed to be positive clone. Compare the amplified DNA of MF to the standard one to check if there is a mutation
12.Digest the pve5233, connect the fragment to DNA of MF, so we have the re-constructed expressing plasmid 13.Transfer the re-constructed plasmid to the competent cell of E.coli
14.Smear the E.coli with re-constructed expressing plasmid on the plate with clarithromycin, select the the normally growing E.coli and do colony PCR. After PCR, the E.coli with production is confirmed to be positive clone. Compare the result to the standard DNA sequence to check if there is a mutation
15.Foster the E.coli with re-constructed expressing plasmid, use Sds-page method to check there is miraculin 16.Collect the plasmid of E.coli with re-constructed plasmid, and transfer the plasmid into the competent cell of lactic acid bacteria
17.Smear the lactic acid bacteria with re-constructed E.coli plasmid on the plate with clarithromycin select the normally growing bacteria and do colony PCR. After PCR, the bacteria with MF’s DNA is confirmed to be positive clone. Then compare the amplified MF’s DNA sequence to the standard sequence to check if there is mutation
18.Foster the lactic acid bacteria with re-constructed plasmid, use Sds-page method to check if there is miraculin expressing
19.The lactic acid bacteria expresses miraculin