Experiments and results

To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the biobricks page. A sketch showing our final construct built from all the necessary biobricks is also given below:

• Bilde

Most of the biobricks we decided to use was already present in the registry, but we also needed biobricks with certain properties that was not present in the registry. These we had to make ourselves. The new bricks we made, that we also characterized, are the following; a protein coding brick for colicin E1, a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, the lld promotor + RBS from E.coli, and the lld promotor + RBS from C.glutamicum.

This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.

Colicin (<partinfo>BBa_K822002</partinfo>)

Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device.

Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)

We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, but the fragments were either not as expected. This is why we thought this biobrick was a good candidate for an improvement. Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).

When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found here. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator is present.

Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:

This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.	Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense.

lld promoter + RBS from E.coli (<partinfo>BBa_K822000</partinfo>)

The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in E.coli into a biobrick, and to use this biobrick in our project http://www.ncbi.nlm.nih.gov/pubmed/18263722 1.

The primers used to amplify the sequence are given below:

The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the E.coli K12 genome sequence provided by the NCBI nucleotide database http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2. These primers in combination with the genome from E.coli K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from E.coli, R because it contains RBS).

We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.

ldhA promoter + RBS from C.glutamicum (<partinfo>BBa_K822001</partinfo>)

We also amplified the ldhA promoter from Corynebacterium glutamicum. This has similar properties as the lld promoter from E.coli, so this promoter was also a candidate to possibly being used as the lactate inducable promoter in our project. The ldhA promoter was amplified using the genome of C.glutamicum ATC 13032 as template, and the primers below:

The promoter has not been properly characterized, but also this was sent to sequencing, and had a 100 % match with the theoretical sequence.