Engineering

Cycle #1 - Gene Fragment Cycle

Taipei-KCISLK-V1 bought samples from a Taiwanese earthworm farm and attempted to extract its total RNA, but we struggled with the extraction process. After research, we realized that the earthworm's diet consisted of soil and nutrients that may have included a lot of unwanted bacteria that could interfere with extraction of lumbrokinase, so we decided to make the earthworms undergo fasting while laying on moisturized tissue containing deionized water. Our experiment focused on taking the earthworms that fasted, and having them dehydrated before performing total RNA extraction as opposed to taking RNA from the earthworms with regular diets. With the help of commercial kits, we aimed to extract the Lumbrokinase gene (BBa_K4673017), in order to perform a quantitative and qualitative analysis on extracted RNA concentration with a spectrophotometer.

Further, the beginning of our RNA extraction method relied on cutting earthworms into pieces and vortexing them, but we realized the quality of extraction was low. Thus, we decided on applying a tissue grinder and glass pestle to homogenize the extracted earthworm tissue, and we also ran gel electrophoresis for further confirmation of Lumbrokinase.

In figure 1, we used the ApE – a plasmid editor to find the mRNA sequence and mature peptide of Lumbrokinase, so that we could confirm our results with the number of base pairs present in Lumbrokinase mRNA.

Cycle #2 - Plasmid Cycle (pGEM-T EASY vector → pET-22b)

To produce Lumbrokinase enzyme using two vectors to solve our issue regarding cloning and preservation, as well as expression. We hypothesize that the pGEM-T EASY vector can be used to efficiently clone and preserve the Lumbrokinase enzyme, while the pET-22b vector can be used to run protein expression. This decision was based on the properties of each of the vectors that made it most suitable for our goals, such as pGEM-T EASY vector’s high copy number, leading to higher enzyme concentrations, and pET-22b vector’s multiple cloning sites and LacZ sequences.

We inserted our PCR fragment into the LacZ gene within the pGEM-T EASY vector, which was in E.coli DH5-alpha (as seen in figure 2), and we determined successful insertion based on blue-white screening. Subsequently, pGEM-T EASY vector’s Lumbrokinase was extracted and purified before insertion into pET-22b vector for transformation into BL21 and protein expression in future steps.

The pGEM-T EASY vector (figure 3) contains multiple cloning sites that are before the RNA promoter, allowing us to insert lumbrokinase in the multiple cloning sites in order for the RNA polymerase to synthesize lumbrokinase.

We designed the insertion of the lumbrokinase gene fragment in the Lumbrokinase_pET-22b vector (figure 4) to be after the lac operator (BBa_K4673024) and before the 6x his-tag (BBa_K4673028) in order for successful synthesis by the RNA polymerase and detection by western blotting in the test cycle, respectively.

Cycle #3 - Primer Design Cycle

Our team encountered issues regarding how to regulate the expression of our lumbrokinase gene, as well as trying to provide a potential solution to aging populations that forget to eat medicine, so our team designed a less used system of protein expression – oleate induction. We chose the presence of fatty acid for our induction expression of lumbrokinase because we want E.coli BL21 to express regularly following the intake of fatty acid, which likely happens daily for individuals, thus achieving regulated daily expression. We designed a primer by substituting the original Lac Operator (BBa_K4673024) with a FadR regulator (BBa_K4673025) on the pET-22b plasmid to create an oleate-induced expression mechanism for the Lumbrokinase gene. We hypothesized that this system will help regulate the expression of Lumbrokinase gene in the presence or absence of oleate in the culture medium.

In the plasmid cycle, we inserted lumbrokinase DNA, which is cut from the pGEM-T EASY vector, into pET-22b vector, and during the insertion process we designed restriction enzymes to cut out the lumbrokinase gene and the Lac operator from pGEM-T EASY vector and the Lac operator and the T7 promoter(BBa_I72074) from pET-22b vector in order to insert the FadR regulator into pET-22b (more details in Dry Lab). After the successful insert of Fad R binding site (figure 5), pFadH_Lumbrokinase_pET-22b (figure 6) has an induction mechanism that has a T7 promoter followed by a FadR regulator.

Cycle #4 - BL21 and Protein Expression Cycle

Only certain strains of E.coli are more suitable for protein expression and we had to determine what kind of parameters are most suitable for expression. Our team performed transformation to E. coli BL21 and we aimed to compare protein expression of Lumbrokinase gene using two altered pET-22b plasmids: one induced by IPTG and the other by oleic acids.

Figure 8 explains the process of IPTG induction and it works similarly to oleate induction in that it relies on the presence of a molecule that binds to the repressor at the promoter region in order to allow for transcription of the genes downstream of the operator. We designed this as it is a common method of recombinant protein production for medication, such as for insulin production.