The Engineering Design Cycle

Throughout the course of our project, we faced many challenges and setbacks we needed to overcome. We also talked to stakeholders and researchers to gain knowledge we otherwise would not have obtained. These warranted repeated changes to our design and tweaks to aspects of our project. Below, we show our iterations through the engineering design cycle, both due to integrated human practices and experimental discoveries.

lfa bunch

Cycle 1


To design the four antibodies required for our project, we BrioBricked four inserts as a composite part. Each composite part has a strong T7 inducible promoter, enabling IPTG induction, an E. coli codon-optimized ribosomal binding site (RBS), the antibody coding sequence, and a terminator. The RBS and antibody coding sequence were designed to serve a different purpose for each antibody. Legal restriction enzymes were also placed between each subcomponent. Next, we decided to use pSB3K3 for our vector, a low-copy number plasmid, which is important to reduce the metabolic burden on our cells.1


We used traditional cloning (TC) incorporate our insert into the pSB3K3 vector in E. coli DH5-alpha cells, however, we received low transformation efficiency. We modified our TC by mixing the digested insert and vector directly in a tube for ligation without performing gel electrophoresis. Recognizing the possibility of our original mRFP gene fragment being re-ligated back into the vector after being digested with restriction enzymes, we screened transformants based on the colonies’ color. Colonies with our insert will appear white, while those with the re-ligated mRFP gene will appear red. After cloning our antibody sequences into DH5-alpha cells and selecting only for white colonies, we utilized restriction mapping and sequencing to verify that the DH5-alpha cells contained our antibody inserts. We then transformed E. coli SHuffle and utilized restriction mapping and sequencing to verify SHuffle contained the correct insert.


To test if our devices expressed our antibodies of interest, we induced SHuffle cells to produce protein and then conducted SDS-PAGE to verify if protein bands were present at the correct molecular weight. However, we observed low expression. Based on this, we changed various parameters of protein induction (incubation time, IPTG concentration, and temperature), but we still did not see significant expression of our target antibodies. See the Protein Induction part of our Results page for further information.


Based on our testing results, review of academic articles, and expert opinions, we learned that other vectors can allow for higher protein expression. Since our pSB3K3 vector is a low copy number plasmid, it may have resulted in too few transcripts being created despite induction.

Cycle 2


We are currently re-designing our constructs so that they use the pET-21b(+) expression vector used by Robinson et al.2, a high copy number plasmid designed for use in T7 induction.

Future Iterations

If modifying these variables does not increase protein yields, we will redesign our BioBrick construct. We will design our RBS to have maximum translational efficiency rather than 75% efficiency and use Robinson promoters to increase protein expression.


Following the Engineering Design Cycle, we were successfully able to build our SHuffle cells and test protein induction.