Engineering Cycle #1

Design

In other peptides and enzymes there are additional amino acids attached to the head and tail of the structure itself. These amino acids are referred to as “g cap” and “poly a tail” which both serve as a protection to the peptide strand itself. This same idea is used when addressing our natural original bombolitin, thus we decided to make three modifications to the bombolitin peptide, and each modification has different amino acids attached to the end. We called the three modifications LLL, FFF, and WWW. The goal is to be able to express these three modifications of bombolitin.

The sequence for the original bombolitin is LNLKKILGKIGVMLSHLN. The unmodified bombolitin has a net charge of +4, hydrophobic ratio of 55%, and GRAVY index of 0.58.

The sequence for the LLL modification is LNLKKILGKIGVMLSHLNLLL. The LLL modification has a net charge of +3.25, hydrophobic ratio of 50%, and GRAVY index of 0.91.

The sequence for the FFF modification is LNLKKILGKIGVMLSHLNFFF. The FFF modification has a net charge of +3.25, hydrophobic ratio of 57%, and GRAVY index of 0.90.

The sequence for the WWW modification is LNLKKILGKIGVMLSHLNWWW. The WWW modification has a net charge of +3.25, hydrophobic ratio of 55%, and GRAVY Index of 0.91.

Build

LLL modification
Forward Primers: F_casette_Bombolitin
Reverse Primers: RLm_casette_Bombolitin

FFF modification
Forward Primers: F_casette_Bombolitin
Reverse Primers: RFm_casette_Bombolitin

WWW modification
Forward Primers: F_casette_Bombolitin
Reverse Primers: RWm_casette_Bombolitin

These three modifications use the same forward primers, but different reverse primers. These modifications all increase the hydrophobicity of bombolitin, which will be able to attract negative biomolecules in the outer membrane of Ralstonia with its increased positive charge from the hydrophobic ratio. Furthermore, the values of the GRAVY Index should be near zero, since it means that the peptide will be able to bind and have antibacterial activity. Thioredoxin was also able to enhance protein expression by decreasing the net charge and hydrophobicity. This makes it so that the bombolitin is not toxic to the host cell and does not kill it after being produced.

Test

When we expressed each modification, we were only able to express the LLL modification successfully. The FFF modification and WWW modifications were not able to be expressed.

Learn

Although we weren't able to express all three modifications of bombolitin, we registered all three as Parts on the Parts Registry Page for other teams to use in the future. The fact that the other two modifications were not able to be expressed could be due to multiple reasons, such as mistakes with the protocol such as annealing the primers at the wrong temperature or using competent cells there were too old during heatshock transformation or electroporation. Furthermore, we also learned about the crucial role of thioredoxin in producing the modified bombolitin since it prevents the competent cells from destroying themselves after producing the modified bombolitin.

EngineeringCycle #2

Design

Ligation is a crucial step in Group 1's work, as it is the segment where the recombinant plasmid is created. The process of ligation requires a vector, an insert, T4 ligase buffer, T4 ligase, and molecular water. Once these have been acquired and mixed together, it is to be incubated for 16 hours at 16°C overnight. In this case, the vector is the pET-32a, and the insert is the modified bombolitin.

Build

After PCR, restriction enzymes BamHI and SalI are used to purify the PCR product. After that, ligation takes place. For this, we used a vector to insert a ratio of 6µL:10µL. For ligation we used 6µL of pET-32a, 10µL of the bombolitin, 2µL of T4 ligase buffer, 0.25µL of T4 ligase, and 1.75µL of molecular water. These are then incubated in the ligation machine for 16 hours at 16°C.

Test

After being incubated in the ligation machine for 16 hours, it is run through gel electrophoresis to check whether or not the modified bombolitin has been successfully inserted into the pET-32a. However, we did not get the expected band size.

Possible Errors

Initially, after we failed for the first few times, we believed that the issue may have lied within our different types of transformation. This was because the first time we worked through the procedure, heatshock transformation showed that the plasmid was present, but as we proceeded through electroporation, the plasmid disappeared. This made us believe that our electroporation was off, but the second time we tried, the plasmid that showed up for heatshock transformation was not of the expected band size either. This implied that the plasmid was just the empty vector, since the band size was smaller than expected, when combined with the insert, the band size should increase.

Size of the bands did not line up with what was expected.

Learn

We believe that we did not get the expected band size because the vector to insert ratio was too high. Due to this, the ratio of the vector to insert was changed to 1:2, where we used 4µL of the vector with 8µL of the insert and 3µL of the vector with 6µL of the insert. With this new ratio, we were able to get the expected band size because we decreased the vector, while keeping the insert the same.

Design

The Group 2 (Kasetsart University) Lab's goal is to determine which nanobodies bind best with the target bacteria: Ralstonia (R.solanacearum). These nanobodies will be used as a target binding antibody in order to fight against bacteria wilt. Through biopanning, the values of the various nanobodies that bind best with the target bacteria will be identified and measured. The results are then checked to ensure whether or not it binds best with the target through indirect ELISA.

• Past pesearch
• The process of biopanning consists of several common steps, including incubating the target material with the library culture, washing unbound bacteria from the material surface, placing the washed material with bound library members into fresh media, and allowing the bound members to amplify [1]. There are, however, some aspects of our dry lab that are unique and advantageous for our project.

Build

In order to determine which nanobody binds best with R.solanacearum bacteria to combat bacterial wilt, biopanning must be performed twice. R.solanacearum will be prepared as a bacteria chain in the form of 96 well plates using a coating buffer that was previously prepared to infect E. coli. The Phosphate-buffered saline solution that will be used contains 0.05% Tween-20, which is less toxic than the alternative, tween-80 and a better fit for the experimental procedures. After determining the values of the antibodies, 20 random colonies will be selected to test for binding capacity through Indirect Enzyme-Linked Immunosorbent Assay (ELISA).

Preparing the Coating Buffer

Test

Biopanning will be completed by immobilizing each prepared bacteria chain onto a well plate with coating buffer. This will be incubated at 37 degrees celsius overnight. The first round of Biopanning will use TG1 E. coli, as it is a suppressor host strain that stops protein synthesis when a stop codon is detected. After that, the detected protein strain will undergo biopanning again, however, this time, using HB2151 E. coli to read the proteins’ codons.

Afterwards, 20 colonies (E.coli HB2151) will be randomly selected to determine binding capacity to R.solanacearum by indirect ELISA. Finally, the binding capacity will be determined through a color indicator where the wells containing the nanobodies will change from blue to yellow.

Learn

From the experiment, the members of the Group 2 Lab learned methods of biopanning and indirect ELISA as well as the multiple trials needed when completing a research lab. Most importantly, they were able to determine the nanobody that binds with R.solanacearum from the experiment.