Team:Uppsala-Sweden/Notebook

Week 1

This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See protocol SOB- medium, LB medium and Competent cell preparation final). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C (See Competent cell preparation final). We received the pTJ122 plasmid carrying the ccaS, ccaR and cph8 genes as well as the PcpcG2 promoter from Christopher A Voigt at University of California San Francisco.

Week 2

2011-06-27

This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 108 transformants /ug DNA.

Blue/green output The strains carrying the amilGFP (green/yellow output) and amilCP (blue output) plasmids were provided by J.F Miller, UCLA. They arrived on plates witch were malhandled during the delivery. Both colors looked really nice but since the colonies were all mixed into each other we started by re-plating to obtain single colonies.

2011-06-28

Blue/green output Started overnight cultures of E coli carrying the plasmids pGEM11- amilGFP and pGEM14- amilCP. We followed protocol Overnight culture and glycerol stock for the initial preparations on the output modules.

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):

pSB1A3-J04450 (ampR backbone) pSB1C3-J04450 (CmR backbone) pSB1K3-J04450 (KanR backbone) pSB1AK3-B0014 (Double terminator) pSB1AK3-B1001 (synthetic terminator) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB1A2-R0011 (PllacO, lacI repressable promotor) pSB2K3-I15010 (cph8 red sensor)

2011-06-29

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):

pSB2K3-Q03530 (cII inverter) pSB3T5-J04450 (low copy vector tetR, ori P15A) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB1A2-K093005 (RBS + RFP, red dye) pSB2K3-I15010 (cph8 red sensor) - had to be redone due to failure in the first attempt.

Followed up by restreaking of the transformants from the previous day.

2011-06-30

Restreaking of the transformants from 11-06-29 and started overnight cultures of the reastreaked plates from the previous day (11-06-28).

2011-07-01

Started with doing overnight cultures from the reastreaked plates from the previous day (11-06-30). After that we prepared 20 % sterile glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.

2011-07-02

All the overnight cultures prepared on the previous day were made into glycerol stock and frozen in -80°C.

Week 3

2011-07-04

Started by doing overnight cultures of the strains carrying these plasmids:

pSB1A3-J04450 (vector, ampR) pSB1C3-J04450 (vector, CmR) pSB1K3-J04450 (vector, KanR) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB2K3-Q03530 (cII inverter) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB2K3-I15010 (cph8 red sensor)

The purpose was to make plasmid preparation the day after.

2011-07-05

The day started of by running a PCR on the DNA template of the green light sensor which includes the two genes ccaR and ccaS as well as the promotor PcpcG2 (see protocol ccaR BioBrick, protocol ccaS BioBrick, protocol PcpcG2 BioBrick).

After lunch we did a plasmid preparation of the overnight cultures from 4/7 (purification protocol). At the end of the day we did a PCR to verify the red light sensor (cph8). We also did site specific mutagensis using PCR for the pigments amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol cph8, protocol amilGFP_EcoRI, protocol amilCP_EcoRI)

2011-07-06

Preparation of agarose gel (1%) for the gel electrophoresis. Then we ran our PCR products from yesterday (11-07-05) in the gel electrophoresis. Finally we initiated the first biobrick assembly session. The following entities were assembled pcyA-RSB, ho1- RSB and Inv.cl-PompC, ho1-gene and RBS.

2011-07-07

re-circularized of pigment vectors after site mutation PCR (Purification protocol, Phosphorylation of DNA, DpnI digestion protocol). Transformation of the ligated plasmids from 11-07-06 (Protocol for transforming TOP10 competent cells). Transformation of mutagenized amilCP and amilGFP. Digestion of ccaS, ccaR and plasmid backbone plasmid pSB1C3 (BioBrick Assembly Manual).

2011-07-08

PCR of EnvZ- Knock out FRT-Kan, Cph8 and pcpcG2 (PCR protocol of envZ knockout FRT-Kan_FRT,...). Transformation of Chp8. Re streak of the transformants from 2011-07-07.

2011-07-10

Today we did Screening of transformants from 11-07-08 (3A assemblies: pcyA-RSB, ho1- RSB and Inv.cl-PompC, ho1-gene and RBS, ccaS and ccaR, amilCP and amilGFP, 6 clones of each, 42 samples in total). We Picked and suspend a colony from each re-streak in 20-30 µl of PBS. The volume depends on the colony size. Small colonies use 15-20 µl, big colonies take 30 µl. We also started overnight culture in selective medium from the suspensions, 1 ml in each. Run colony PCR from of each suspended colony using taq polymerase. Last thing was run an agarose gel of PCR products.

Week 4

2011-07-11

Today we did a frozen stock of all overnight cultures in -80°C freezer (ccaS clone 1,RBS-pcyA, amilGFP, amilCP, ccaR, RBS-ho1, PompC-Inv.cI). We also started 3 ml cultures by inoculating them with 100 µl of the overnight cultures of the two clones of ccaR, RBS-pcyA and RBS-ho1, the constructs needed for the upcoming assemblies. When the cultures had grown for about 4 hours, we did plasmid preparations of them. We did PCR of amilCP using the pGEM14-amilCP_EcRI plasmid as template and using primers to add BioBrick prefix and suffix. Mutagensis of amilGFP to remove PstI site and ccaS_EcoRI mutagenesis, round one.

After studying the experience section on the Registry’s homepage, we decided to change inverters to parts Q04400 (TetR inverter) and Q04510 (cI inverter). The inverters we originally chose were not so well characterized, and the promoters they used as output might be too weak for our system.

Transformation of parts Q04400 (TetR inverter) and Q04510 (cI inverter).

2011-07-12

The day started by running a gel of the mutagenized ccaS, amilCP-BB10 PCR product, and amilGFP_PstI linear plasmid. At the same time we prepared the samples (ccaR, ccaS, RBS-ho1 and RBS-pcyA) for sequencing. Plating of strains carrying plasmids with terminator B1001, PLlacO, pSB3T5 and pSB4K5. Purification of PCR products of ccaS_EcoRI, amilGFP_PstI, amilCP_BB10, and PcpcG2. Re-ligation of mutagenized ccaS and amilGFP, and cloning of amilCP_BB10.

The transformation of inverters from yesterday didn’t go well. The lambda-cI-inverter had only a few colonies, while the tetR inverter had no colonies at all. So TetR inverter has to be redone. This time the transformation used 2 µl of DNA from the MIT distribution. Re-streak of cI inverter transformants from 2011-07-11.

2011-07-13

We started the day by running EnvZ-KO, ccaS, EcoRI- digested amilCP and amilGFP on a gel. We obtained good results; there were no distinguishable multiple bands on the gel, although the bands seemed a bit smeared. Then we did transformation of ccaS_EcoRI, amilGFP_PstI, pSB1A3-amilCP, pSB-pcpcG2 and pSB4A5 backbone. Re-streak (There were one colony on the whole plate) and colony PCR of tetR inverter (DNA taken from well distributed from MIT).

Started overnight cultures of B1001 terminator, PlacO, PSB3T5 and PSB4K5-backbone (prepared for frozen stock + plasmid preparation) and finally RFP with RBS and lambda c1.inv (prepared for frozen stock + plasmid preparation).

The last thing we did was to make new plates of all the three antibiotics (A, C, K). We also made some plate with tetracycline.

2011-07-14

Re-streak pSB1C3-ccaS_EcoRI, pGEM-amilGFP and pSB4A5. We were supposed to re-streak pSB1A3-amilCP and pSB1A3-PcpcG2 as well, but the transformants had no colonies at all. Suspect wrong plasmid backbone or no ligase added during the cloning attempt. To investigate this we did a transformation of the plasmid backbone (PSB1A3) and plated it on plates containing antibiotics ampicillin, chloramphenicol and kanamycin. We did plasmid prep of pSB1AK3-B1001, pSB1A2-R0011, pSB3T5-backbone, pSB4K5 backbone. New agar plates with antibiotics ampicillin, chloramphenicol and kanamycin, just made in the morning. Assembly of chromophores, step Cloning of tetR inverter into new backbone. Cloning of pSB1A3-amilCP and pSB1A3-PcpcG2 again. Overnight culture of pSB1A3 from the strain collection to test whether it’s right.

The sequencing result arrived today around lunch time! RBS-pcyA strains we sent for sequencing were good. ccaR clone number 2 is better than clone number 1. Clone 1 of RBS-ho1 will be used in further assembly steps.