Difference between revisions of "Part:BBa K123002"

Revision as of 16:50, 21 October 2021

LacIQ ERE TetR

This composite part contains LacIQ (BBa_I14032) and Estrogen Receptor Element (ERE) and TetR (BBa_C0040). The Estrogen Responsive element is to be used in conjunction with ER (Estrogen Receptor). This design involves ER binding to the ERE and disrupting the constitutive promoter LacIQ from producing TetR via physically getting in the way of the polymerase.

Improvement of ERE controlled TetR Expression - Alma 2021

When we thought we had succeeded in improving K123002, we sent 2 colonies for sequencing and received the following results.

This is proof that K3737004 contains the stronger promoter, Part BBa_J23100.

This is proof that K3737004 contains the human estrogen receptor element, part BBa_K3737003.

This comparison of K3737004 and ribosome binding site (RBS) shows that no significant similarity was found between the two sequences. Although this indicates that K3737004 does not contain the RBS like we expected, we have improved upon K123002 in other ways and are still working to include the RBS.

When comparing K123002 to K3737004 via qPCR, we used the difference between CT values of the strains with and without reverse transcriptase (RT) to calculate the amount of RNA present. We used two clones of K3737004; both clones showed lower CT values with RT than without. This proves that more DNA was made from the RNA present in clones with RT compared to the clones without RT. K123002 had very similar CT values. This means that converting the RNA present did not make any more DNA, and there was no transcription of TetR in the K123002 part. Because we used tet_seqR and tet_seqF primers, we know that the TetR gene was present in K3737004 and not present in K123002.

The amplification plot made from the qPCR is a visual representation of our data. We also made amplification plots to show the difference in amplification between K123002 and K3737004.

The dissociation curve shows only one peak, proving that there is only 1 type of DNA in the qPCR samples. This proves that the tet_seqR and tet_seqF were the only primers that were able to bind any genes in the samples. This is promising because it indicates that clone 1 and clone 2 are the same sequence. Based on this data, we have successfully improved upon K123002.

File:Alma qpcrdissociation Sequence and Features