Team:Uppsala-SwedenWeek10

From 2010.igem.org

Week-10

Construction of G1- G6

5 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.

Figure 3

Again, inoculated samples were sent to perform plasmid extraction and measure the plasmid concentration.

Plasmids were digested with proper enzymes bought from Sigma-Aldrich and run for gel to confirm the size.

Figure 4 Gel picture shows the length of digested G sets

From the gel, we think G1 and G2C4 seems having the right biobricks in size. G5 seems not digested well, though the plasmid seems in right size. The colonies we picked for G3, G4, G6 seems in wrong size according to c-PCR. Another transformation is necessary for G3, G4, and G6 and the starting biobricks need to be checked again by gel.

Figure 5 Gel result to verify length of G5C3 and the starting material of G3, G4, and G6

All the starting material of G3, G4, and G6 seems good, though G5 C3 doesn’t seems in the right length. So a second transformation was done for G3, G4, G5, G6. After transformation, the cells were plated on the proper antibiotic plates. 12 colonies were picked from each plates and sent to perform c-PCR. The c-PCR result was shown on the gel picture.

Figure 6 Gel picture shows the c-PCR result of second time transformation product of G3. The samples marked in orange were thought in right size and proceed to inoculate

Figure 7 Gel picture shows the c-PCR result of second time transformation product of G4. The samples marked in orange were thought in right size and proceed to inoculate.

Figure 8 Gel picture shows the c-PCR result of second time transformation product of G5. The samples marked in orange were thought in right size and proceed to inoculate.

Figure 9 Gel picture shows the c-PCR result of second time transformation product of G6. The samples marked in orange were thought in right size and proceed to inoculate.

Selected colonies were inoculated and performed plasmid extraction on the second day.

For further confirm the construction of G sets, we decided to use some specific digestion and run the gel to verify the special sites on each construct. The digestion enzymes were chosen by using Gentle.

Specific Digestion Enzymes G3 BglI EcoRV PstI G4 Eco31I BglI EcoRI PstI G5 HinII EcoRV EcoRI PstI G6 EcoRI PstI BglII

Table . Specific enzymes chosen for each construct and the expected length of all digested parts

Figure 10 Gel picture shows the result of specific digestion of G3, G4, G5 and G6.

Only samples from G4 and G6 showed in the expected length and consistence. A second specific digestion were performed to samples of G3 and G5

Figure 11 Gel picture shows the result of specifc digestion of G3 and G5

Comparing the expected length of digested parts, G3C5, G4C6, G5C12 and G6C6 were digested and ligated according to the plan