May

Design considerations

A promoter of the Anderson constitutive promoter family was placed in front of the riboswitch. It is the consensus constitutive promoter family of iGEM, it is well documented and the amount of expression differs 2500 fold between different promoters. It can also be easily swapped for another promoter of the same family if a different amount of expression is needed⁷. This is convenient since the optimal expression level of the toxin gene is not known yet.

02 - 12 May

First attempt in constructing the inverter part out of biobricks Bba_P0140 and Bba_I13521. This has been done via the standard iGEM 3A assembly protocol [Link]. The transformation resulted in one red colony.

This colony has been checked by placing the constitutive promoter J23119 in front of it via another 3A assembly. If the construct is correct, the result should be white colonies.

However, this was not the case. Sequencing and PCR results showed that the construct only contained Bba_P0140.

13 - 24 May

Creation of the riboswitch part out of Bascillis subtilis and Escherichia coli. The primers used for this can be found in table X. Extraction of genomic DNA from B. subtilis and E. coli was done with the use of the GeneJet genomic DNA purification kit and an overnight culture of B. subtilis strain 168 that has grown in B. subtilis medium and an overnight culture of E. coli strain DH5𝛂 in normal LB medium. The extraction was done according to the supplied Gram-positive and Gram-negative bacteria standard protocol.

For the PCR the standard protocol of New England Biolab has been used. For the retrieval of the vitamin b12 riboswitch out of Escherichia coli DH5alpha an annealing temperature of 66° was used and for the guanine riboswitch out of Bascillis subtilis an annealing temperature of 63°. PCR and sequencing results showed that both constructs were correctly retrieved.

Via 3A-assembly the riboswitch part and the inverter part have been assembled. This resulted in three types of colonies: white, pink and red. The correct construct should be around 2500 base pairs.

PCR amplification and sequencing results showed that the pink colonies where the correct construct.

05 - 20 July

To provide a basic proof of principle of the designed construct, a colony containing the B/GRinv plasmid was grown overnight in 10 mL LB medium. The concentration range of guanine to test the guanine riboswitch construct were based on the concentrations used in previous experiments with guanine riboswitches from B. Subtilis. The concentration range of vitamin b12 was based on the concentrations normally used for testing bacteria that are used to determine the amount of vitamin b12 in human blood. Five tubes containing a concentration of f 0, 200 ng L-1 , 2 μg L-1, 20 μg L-1, 200 μg L-1 and 2 mg L-1 vitamin b12 and 0, 0.01, 0.05, 0.1, 0.5 and 1 mg mL-1 guanine were used. These cultures were spin down at 4700 RPM for 3 minutes in order to compare the colouration of the pellets.

05 September - 05 October

For the second proof of principle experiment, agar plates were overgrown with E. coli containing the B/GRinv. In the middle of the plate a small scoop (0,5 cm in diameter) was taken out and filled with 100 µL of a 50 mg mL-1 guanine solution or 100 µL of a 200 mL L-1 vitamin b12 solution to create a decreasing gradient from the middle. To provide clear images showing the expressed mRFP, pictures were taking using the G:Box gel imager of Syngene. The RFP filter protocol provided with the software programme was used.