Engineering

Overview

Cloning
Transforming B. subtilis
Assays
Future Experiments and Directions

Cloning

We succeeded in cloning and verifying the sequences of all of our Basic parts in the Parts Registry and all of the Composite parts with the exception of the parts with bcaP and bkdB under the control of Pveg, the constitutive promoter.

We tried a number of methods to clone bcaP and bkdB:

1) Golden Gate Assembly with NEB 5 alpha comp cells

We had very low efficiency and the plasmids we extracted were missing various components even after a few iterations. One mentor suggested that bcaP and bkdB may be toxic to E. coli, as bcaP is a periplasmic membrane protein and E. coli is a gram negative bacteria with no periplasm and bkdB is one subunit of the larger BCKDH complex. We wanted to investigate this hypothesis, so we decided to try assembling the construct with an empty promoter and rbs to see if stopping expression of the genes completely would make a difference.

2) Golden Gate Assembly with pL0.p (an empty promoter) and pL0.u (an empty rbs)

If these constructs were assembled successfully, it would prove that the reason for the continued failures might be because expression of bcaP and bkdB is toxic to E. coli. We were able to successfully clone a bcaP construct with an empty rbs site, confirming bcaP and bkdB are likely toxic to E. coli. Next, we tried down regulating expression of these genes with a different promoter.

3) Golden Gate Assembly with AckA (a promoter from B. subtilis that is nonfunctional/has low expression in E. coli)

We had one other promoter in our inventory aside from Pveg and AckA. This promoter is inhibited by CodY, a regulatory protein that is responsive to BCAA levels in B. subtilis. As we cloned mApple2 into E. coli we saw that mApple2 under constitutive control by Pveg was expressed more strongly in E. coli than mApple2 under control by AckA. We hoped a down regulated promoter would prevent toxicity; however, the constructs were still missing various pieces.

4) Golden Gate Assembly with Pveg using NEB stable cells

One mentor suggested using stable cells. These are competent E. coli cells that are well suited for cloning unstable inserts or sequences with direct or inverted repeats. We transformed our constructs with Pveg into stable cells. We attempted to grow our transformants at 37 °C, 30 °C, and room temperature (~25-27 °C). When left at room temperature for ~1-2 days, the plates with bcaP and bkdB had a lot of growth, similar to other constructs after growing at 37 °C for 16 hours. We inoculated several colonies and shook them at room temperature. Only a few inoculations had significant enough growth to miniprep, and the constructs were still faulty. However, given the number of independent colonies that grew, the possibility of a correct construct remains high. Given enough time, a colony with the correct sequence could be selected and validated.

5) Golden Gate Assembly with Ppen (a promoter that may only work in B. subtilis)

Another mentor suggested that we could look for promoters that only work in B. subtilis and not E. coli. We looked into Ppen, a strong, constitutive promoter in B. subtilis. The Grossman Lab uses Ppen to express LacZ for blue white screening in B. subtilis. However, this construct does not display a blue phenotype in E. coli. Our mentor concluded that Ppen might not work in E. coli or drives very low expression of the gene it controls. We are still in the process of cloning Ppen into the appropriate Composite parts, so we will not have time to test this construct before the Jamboree.

6) Golden Gate Assembly using an inverted Pveg promoter

We originally designed this inverted Pveg promoter so Cre recombinase could turn on constitutive expression of bcaP and ilvE/ilvK when we induced the system with doxycycline. The Pveg promoter is essentially nonfunctional when inverted. We expected we could clone the construct successfully. We were able to successfully clone this construct into a pL2 Composite part and verify its sequence! We plan on using this construct in conjunction with Cre for future assays, although there may still be some leakiness since our transformed cells grew relatively slowly.

Transforming B. subtilis

We have succeeded in transforming all of the pL2 Composite parts with pSB_AMY and pSB_AZL backbones.

Our team had significant experience transforming E. coli because we used E. coli as the vector for cloning our plasmids, however, we had very limited knowledge of how to transform B. subtilis as it requires different steps for making competent cells and performing the transformation (See more about our protocols on the !)

Initially, we made our own resuspension media and our own competent cells. We asked our graduate mentor in the Grossman Lab for control genomic DNA (gDNA) with various antibiotic resistance genes to try transforming B. subtilis with; however, efficiency was not high. The same happened when we tried transforming our pL2 constructs as well.

We worked with our mentor, trying various combinations of media, plates, volumes of DNA, and PCR versus digest DNA. Ultimately we learned that DNA amplified with PCR generally had higher efficiency in comparison to inserts digested from pL2 plasmids. In addition, we learned that fresh competent cells are better than freezer strains of competent cells.

The first row of plates show the transformation of BBa_K4074050 (Constitutive mApple with Kan Selection) using 1) 5 uL of PCR product with our mentor’s media, 2) 10 uL of PCR product with our mentor’s media, 3) 15 uL of PCR product with our media. The second row of plates shows the transformation of control DNA with mApple using 1) 3 uL of gDNA and our media and 2) 1 uL of gDNA and our mentor’s media. Not pictured are B. subtilis transformed with digested DNA. Colonies grew, but efficiency was quite low. From these results, we decided to proceed with using our mentor’s media and transform using 10 uL of PCR product and 10 uL of digested DNA in parallel.

Assays

In order to measure rates of BCAA uptake in B. subtilis, we used a BCAA colorimetric assay kit from Sigma Aldrich.

We designed our own protocols for the assay. First, we tested the sensitivity of the kit to a range of concentrations and constructed a standard curve. Following this, we tested different combinations of BCAAs to see if the standard curves generated by these combinations were consistent.

Next, we tested detection of baseline BCAA levels in wild type B. subtilis. In our first iteration of this assay, we grew B. subtilis in LB media; however, we quickly discovered that the BCAA content in LB is too high and created unwanted noise. We then contacted one of our mentors about growing B. subtilis in BCAA-free minimal media. We designed a second experiment using minimal media, and obtained reads in a reasonable range.

Finally, we wanted to conduct a time point assay, measuring BCAA concentrations in liquid culture with B. subtilis over the spam of several hours. This would allow us to get a baseline idea of how effective B. subtilis is at uptaking BCAA’s from the surrounding environment, as our goal is to optimize this process. In our first iteration of this experiment, we started with 400 uM of BCAAs in culture; this was on the lower range of concentrations that our plate reader can detect, and created noise. For our next two experiments, we decided to increase the starting concentration of BCAAs to 800 uM and also run experiments in duplicate, as our mentors suggested. After noticing the results did not show a significant change in BCAA levels, we decided to grow B. subtilis for a longer time to observe BCAA reduction in the media during longer periods of growth.

Read about our results on the Results page!

Future Experiments and Directions

Recombinase-based switch mechanism
We have constructed Cre in a pL2 with tetR in a multi-gene Composite part. Given enough time, we would test the expression of Cre in B. subtilis using SDS-PAGE. Using the bcaP construct with the inverted Pveg promoter, we would also test expression of bcaP, since we expect overexpression of this importer to increase the rate of uptake of BCAAs in B. subtilis significantly.

In addition, we have a construct of mApple2 with loxP sites surrounding it that we successfully transformed into B. subtilis. Once we are able to express the Cre and tetR Composite part in B. subtilis, we plan to induce the system with dox, extract the gDNA of B. subtilis, and perform colony PCR or sequence the insert at various time intervals to measure how long it takes Cre to excise the gene. Based on this timeframe, we could also measure when Cre inverts the reversed Pveg promoter on the constructs relevant to our project and see if this makes a noticeable difference in the amount of BCAAs uptaken by B. subtilis after adding dox.

Transforming and testing multiple parts
While we only had time to transform B. subtilis with one pL2 at a time, we would also like to transform all the pL2’s into each of the different loci in B. subtilis and test how combining these different parts affects the rate of BCAA uptake.