Main purpose of all constructs:
All of the constructs contain these genes: The pelB leader sequence, mature CDA2 gene, and the eGFP gene. The pelB leader sequence is necessary because it tags the CDA gene and leads it into the periplasm. The expression of the CDA gene in the periplasm will be useful for extracting the protein. The eGFP will allow us to physically see that the DNA constructs were successfully transcribed and translated. The CDA protein produced will then be available to use to break down chitin and convert it into chitosan which has many industrial applications.
Purification and use:
The constructs were ordered online at IDT and they came as gBlocks. (each construct contained 1000 ng). The constructs were resuspended as the instructions that came with the gBlocks stated. Then, they were digested (to eliminate the biobricks) and ligated into pSB1C3 and pSB1T3 plasmids. Once they were ligated into the plasmids, they were transformed into competent E. coli cells so that the genes may be expressed.
Construct 1: Bicistronic (BBa_K1662000)
The bicistronic construct consists of two promoters: the TetR promoter and the P(Lac) IQ promoter. This construct creates two different mRNA transcripts. One transcript will consist of the pelB leader sequence and the mature CDA2 gene which are both expressed under the TetR promoter while the other transcript will have the eGFP gene which is expressed under the P(Lac)IQ promoter. The eGFP will remain in the cytoplasm while the pelB leader sequence will lead the mature CDA2 gene into the periplasm. This means that the entire E. coli will glow green if the DNA construct is transcribed and translated correctly.
Construct 2: Bidirectional (BBa_K1662002)
The bidirectional construct only uses one promoter: the TetR promoter. This serves as an advantage because unlike the bicistronic construct, only one antibiotic (tetracycline) is needed to control gene expression. The construct puts two TetR promoters back to back with a multiple cloning site in between them. One TetR promoter controls the pelB + mature CDA2 genes while the other controls the eGFP gene. Once again, this creates two separate mRNA transcripts and the eGFP will be expressed in the cytoplasm while the mature CDA2 gene is led into the periplasm by the pelB leader sequence, just like the bicistronic construct.
Construct 3: Fusion (BBaK1662002)
The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.