Team:UChile-OpenBio/Results
En construcción
1.- Gibson Assembly d-LDH:
We performed the first attempt of gibson assembly using the LDH_1 (618 bp) and LDH_2 (636 bp) calcutaling the pmol needed for proper ratio of each one, using the equation of New England Biolabs protocol.
pmol = 50 ng/ uL x 1000 / 618 bp x 650 = 50.000 / 401.700 = 0.124 pmol/uL
pmol = 50 ng/uL x 1000 /635 bp x 650 = 50.000 / 412.750 = 0.121 pmol/uL
The figure 1 indicates that the reaction did work but at very low concentration of DNA. We must purify the band and then reamplify using primer suffix an prefix PxSx fwd and rev.
Figure 1. Agarose electrophoresis 1% with 4 uL Gelred DNA staining.
2.- Digestion and ligation of cloning fragments SegA,pLux, p_rfcB, p_BAD and pC.
The digestion and ligation of the 5 biobricks SegA, p_lux, p_rfcB, p_BAD and p_C.
The plasmid backbone and the insert are digested using EcoRI and PstI at 37°C x 30 min, and then ligation is performed overnight at 16°C. Next day chemocompetents cells are a transformed using 2 uL of ligation mix and cultivated agar plates with Ampicillin 100 ug/mL, Chloramphenicol 25 ug/mL and Kanamycin 50 ug/mL correspondingly to prfcB and pBAD in pSB1A3; pC and pLux in pSB1K3 and SegA in pSB1C3. Figure 2 shows one of the transformations in plate using 50 and 100 uL of cell culture. There are no transformation observed. So then we used a large amount of 5 uL of ligation mix and repeated transformation. Figure 3 shows the efficiente transformatoin of prfcB, the only transformation that did work (other data no shown). The other transformation for SegA, pLux, prfcB, pBAD and pC did not have might not have sufficient concentration of inserts. So the, the ligation must be repeated using higher quantiities of insert.
Figure 2. Transformation with prfcB using 50 and 100 uL of transformed cells.
Figure 3. Transformation II (5 uL of ligation mix) with prfcB using 50 and 100 uL of transformed cells.
3.- Gibson Assembly p-CoA-T1 and T2
The construction of p-CoA-T2 from C. necator is assembled by using the Gibson assembly of gBlocks. Figure 4 shows the gibson assembly reactions of pCoAT1_1 and pCoAT1_2 (in lane 1), pCoAT1_3 and pCoAT1_4 (in lane 2) and pCoAT2_3 and pCoAT2_4 (lane T2). The results indicate no assembly of all samples. We inquired the information of the quantities specified in each gBlock tube from the manufacturer. Unfortunately we found that the concentration is lower than the specified and could be the reason why the modules does not worth. This conclude that is imperative to re-amplify gBlocks to perform higher concentrations of isothermal reactions.
Figure 4. Gibson assembly de pCoA-T1 y T2.
4.- PCR colonia (pendiente para 7 Agosto)
We must verify the make the diagnosis of transformed colonies identified in the plates by a Go Taq Protocol colony PCR.
Figure 5. Plates of transformation of prfcB an LuxR. The colonies are selected in randomdly.
Figure 6. Lane 1-10: colonies prfcB. Lane 10-20: colonies LuxR
Figure 2. Transformation with prfcB using 50 and 100 uL of transformed cells.
Figure 3. Transformation II (5 uL of ligation mix) with prfcB using 50 and 100 uL of transformed cells.
3.- Gibson Assembly p-CoA-T1 and T2
The construction of p-CoA-T2 from C. necator is assembled by using the Gibson assembly of gBlocks. Figure 4 shows the gibson assembly reactions of pCoAT1_1 and pCoAT1_2 (in lane 1), pCoAT1_3 and pCoAT1_4 (in lane 2) and pCoAT2_3 and pCoAT2_4 (lane T2). The results indicate no assembly of all samples. We inquired the information of the quantities specified in each gBlock tube from the manufacturer. Unfortunately we found that the concentration is lower than the specified and could be the reason why the modules does not worth. This conclude that is imperative to re-amplify gBlocks to perform higher concentrations of isothermal reactions.
Figure 4. Gibson assembly de pCoA-T1 y T2.
4.- PCR colonia (pendiente para 7 Agosto)
We must verify the make the diagnosis of transformed colonies identified in the plates by a Go Taq Protocol colony PCR.
Figure 5. Plates of transformation of prfcB an LuxR. The colonies are selected in randomdly.
Figure 6. Lane 1-10: colonies prfcB. Lane 10-20: colonies LuxR