Difference between revisions of "Template:Groningen/Labjournal/nucA-in-pSB1C3"

Obtaining RBS (BBa_B0030) and nucA (BBa_K729004)

Experiment:

01/08/16: The RBS (BBa_B0030) was obtained from the part distribution 2016, the plasmid was transformed to E. coli Top10 using this protocol. The nucA (BBa_K729004) was requested from the iGEM headquarters. Colonies from these transformations were cultured in LB medium. Grown cultures of E. coli Top10 with RBS and nucA were used to obtain a glycerol stocks and plasmid isolation was performed (QIAprep® Spin Miniprep Kit). The concentration of the plasmids obtained was measured on Nanodrop. The plasmids were stored at -20°C.

Restriction digestion

Experiment:

28/09/16: On this day restriction digestion of RBS (BBa_B0030) and nucA (BBa_K729004) was performed. The backbone RBS (BBa_B0030) was digested with SpeI and PstI. The insert nucA was digested with XbaI and PstI.

RD mixture:

20 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

The digestion mixture of backbone RBS in pSB1C3 and insert nucA was loaded on a gel to extract both parts. For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

The digestion was successful because the band of the nucA could be seen: 500 bp and RBS in pSB1C3 showed a band of 2,000 bp.

Procedure after gel validation:

Digested nucA and RBS in pSB1C3 were cut out from the gel and DNA was extracted by (Agarose Gel Extraction Kit – Jena Bioscience).

Ligation

Experiment:

28/09/16: The cut nucA was ligated to the SpeI and PstI cut RBS in pSB1C3.

Ligation mixture:

20 μl ligation assay was performed according to the following protocol.

Transformation

Experiment:

28/09/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the transformation protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct constructs. The next day colonies were picked to perform colony PCR to find the correct constructs with the primers VF2 and VR. Find primers here.

PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:

DNA electrophoresis:

For detailed information on how to prepare and run agarose gel see the following protocol.

Conclusion:

Transformation of RBS+nucA in pSB1C3 in E. coli Top10 appeared to be successful. The samples 2, 5, 6 and 7 showed the right size of band. These samples were used to obtain the plasmid from an overnight culture.

Validation

Experiment:

29/09/16: Grown cultures of E. coli Top10 with RBS+nucA in pSB1C3 were used to obtain the glycerol stocks and plasmid isolation was performed (QIAprep® Spin Miniprep Kit). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

Plasmids RBS+nucA in pSB1C3 from colonies 2, 5, and 7 were sent for sequencing with the sequencing primers VF2 and VR. (See primer list).

Conclusion:

Sequencing results showed that the nucA was cloned in the pSB1C3 backbone but the RBS was missing and the suffix was also incorrect. See Figure 4. The nucA (BBa_K729004) received from the iGEM headquarters was sent for sequencing as well. It turned out that the part BBa_K729004 does have the nucA gene but the prefix and suffix are incorrect . See sequencing results in Figure 5 to 7.

Experiments

We wanted to improve the BBa_K729004 part by giving it the correct prefix and suffix. Unfortunately we did not succeed to complete this task.