Aim of experiment: To set up transformations of pQE80-L-LS into M15pREP4 E.coli

Protocols Used: Transformations

Results: N/A

Next Steps: If the transformations are successful, colonies will be selected for overnight cultures tomorrow.

Aim of experiment: To set up overnight cultures of M15pREP4 E.coli containing pQE80-L-LS.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: The overnight cultures will be miniprepped tomorrow.

Aim of experiment: To miniprep the M15pREP4 E.coli Overnight Cultures containing pQE80-L-LS.

Protocols Used: Plasmid Purification

Results: N/A

Next Steps: The miniprep with highest concentration will be sent for sequencing.

Aim of experiment: To amplify LS for cloning into pSB1C3.

Protocols Used: PCR

Results: Figure 2

Next Steps: The gel extracted PCR product will be digested tomorrow and ligations with pSB1C3 will be set up.

Aim of experiment: To digest the LS PCR product with EcoRI and PstI and then set up ligations with pSB1C3.

Protocols Used: Restriction Digests Ligations

Results: N/A

Next Steps: Ligations will be left over the weekend and transformed into JM110 E.coli on Monday.

Aim of experiment: To transform pSB1C3-LS into JM110 E.coli.

Protocols Used: Transformations

Results:N/A

Next Steps:If transformations are successful, overnight cultures will be set up tomorrow.

Aim of experiment: To set up overnight cultures of JM110 E.coli containing pSB1C3-LS from the transformations carried out yesterday.

Protocols Used: Overnight Cultures

Results: N/A

Next Steps: The overnight cultures will be miniprepped and sent for sequencing tomorrow.

Aim of experiment: To miniprep the JM110 E.coli Overnight Cultures containing pSB1C3-LS.

Protocols Used:

Results: N/A

Next Steps: The miniprep with the highest concentration will be sent for sequencing to confirm presence of insert. Sequencing for pQE80-L-LS came back correct so overexpression assays will be started tomorrow.

Aim of experiment: To set up overexpression assays of LS using different concentrations of IPTG.

Protocols Used: Overexpression Assays Note: Cultures were induced with 0 mM, 0.5 mM, 1 mM and 2 mM IPTG. The membrane for the blot was left blocking overnight.

Results: Figure 3

Next Steps: The gel shows a band corresponding to the expected size of LS. However, Western Blotting will confirm if it is indeed LS.

Aim of experiment: To complete characterisation of LS via Western Blotting.

Protocols Used: Westen Blotting

Results: Figure 4

Next Steps: The blot shows that LS has been successfully detected at the expected size of 80 kDa. There is also partial degradation of the protein. Nevertheless, purification of LS will be started next week.

Aim of Experiment: To set up overnight cultures of M15pREP4 E.coli containing pQE80-L-LS.

Protocols Used: Overnight Cultures Note: 150 ml cultures were set up.

Results: N/A

Next Steps: The overnight cultures will be used to innoculate a 3 L day culture tomorrow.

Aim of experiment: To set up 3 L day cultures of M15pREP4 E.coli containing pQE80-L-LS.

Protocols Used: Protein Purification Note: The protocol was stopped at Step 6- protein purification will be continued tomorrow.

Results: N/A

Next Steps: Affinity chromatography will be carried out tomorrow.

Aim of experiment: To continue purification of LS.

Protocols Used: Protein Purification Note: Protocol was stopped at Step 13.

Results: Figure 5 Figure 6

Next Steps: The SDS gel suggests that the peak observed in the affinity chromatography step may due to a smaller sized protein which has shown up strongly on the gel. However, there is still a band observable around the 75 kDa marker which may be LS. The samples will be pooled together and concentrated. A small sample will be blotted and then size exclusion chromaography can be performed.