Team:UC Chile/Cyano/Labook/may

May

Monthly Summary

As Gibson assemblies are not working properly, constructs are being prepared in parallel by Standard Biobrick Assembly. After many attempts we concluded that the enzymes in the master mix are defective and new ones were ordered. There will be no Gibson attempts until arrival.

Digestion and Ligation attempts

pPMQAK1 (expression plasmid) was miniprepped and transformed.

The plan was to get ccab toxin out of the plasmid for further use.

1st attempt: negative control (pPMQAK1 with toxin) was positive.

Comments: possible reasons? Contamination, other resistance, toxin is not toxic. Plasmid sent is not pPMQAK1.

pPMQAK1 was digested.

Colony PCR for C1/2, C1/2 -, C1

New primers arrived:

New PCR attempts and new transformations.

Started to assemble constructs containing Lux, GFP and RFP.

Standard biobrick assembly was attempted (x2 times). There were no colonies in transformation.

Gibson Assembly Attempts

PCR for Kanr was repeated.

F. Federici suggested a way to assemble constructs using psB4K5 (steps must be detailed).

Digestion of double terminator (P1003+B0012+B0014) was done to verify assembly.

Transformation according to Federici’s suggestion: there were no colonies in negative control.

One last big PCR purification to attempt assembly of C1 and C1/2. Gibson assembly and transformation.

Results: no colonies.

A Gibson assembly with sfGFP and December’s master mix (we know it works) was done in order to check the efficiency of the method. After transformation no colonies were observed.

Comments: Gibson current master mix could be wrong.

Restriction and analytic digestion of P1003, B0014 and psB1C3 revealed correct parts size (these will be used for a new Gibson). New Gibson Master Mix prepared. Bacteria with new Master Mix: no colonies.

Another possibly reason for the unsuccess of Gibson assembly could be that the T5 enzyme chews back an T5 enzyme chews back an important amount of base pairs and would be detrimental for the parts with small length.

An experiment trying Gibson assembly with different T5 concentrations was done. Best yield at: +10 ^ (1/2)x

Cell competence was measured: 2.5x10^5 (low)

Attempted a Gibson with all possible parts. No colonies and no colonies in positive control were observed after transformation. Comments: we believe that the enzymes for the reaction are defective. New enzymes were ordered. No new Gibson assemblies will be attempted until the arrival.