Team:Exeter/lab book/1gp/wk1

Operon Construction  |  Glycobase

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Wednesday 11th July (14.30)

• BioBrick extraction of pBAD/AraC promoter weak (BBa_K206001), pBAD/AraC promoter strong (BBa_K206000), pLacI/Ara-1 promoter (BBa_K094120) and a (double) terminator (BBa_B0014)

• Transformation of pBAD/AraC promoter weak, pBAD/AraC promoter strong, pLacI/Ara-1 promoter and terminator

2 x 25µL and 1 x 50µL of One Shot® competent cells were used instead of 3 x 50µL for all transformations.

Thursday 12th July (15.00)

• Adding cultures with pBAD/AraC promoter weak, pBAD/AraC promoter strong, pLacI/Ara-1 promoter and terminator into liquid medium and incubation overnight

Protocol was followed using ampicillin for all BioBrick parts and kanamycin for the terminator as selection antibiotics.

Friday 13th July (9.00)

• Mini-Prepping of pBAD/AraC promoter weak, pBAD/AraC promoter strong, pLacI/Ara-1 promoter and terminator

• Gel Electrophoresis to check fragment sizes of pBAD/AraC promoter weak, pBAD/AraC promoter strong, pLacI/Ara-1 promoter and terminator

Protocol for Mini-Prep was followed exactly, except:

o For centrifugation of 5mL culture at 3'900rcf for 2 minutes at 21°C prior to the purification protocol.

o Prior to step 4, each Falcon tube containing Lysis and Neutralization Solution in addition to the lysate was transferred to fresh 1.5mL Eppendorf tubes so spinning down would be more effective, conducted at 16'100rcf for 5 minutes at 21°C.

o 850µL of the supernatant was withdrawn (being careful not to disturb the cellular debris) and transferred to a geneJET Miniprep column at step 5.

o The geneJET Miniprep column was then centrifuged at 16'100rcf for 1 minute at 21°C at step 6.

o Centrifugation at step 7, 8 and 9 was conducted at 16'100rcf for 1 minute at 21°C.

o MilliQ H2O (50µL) was added to each Eppendorf and left for a couple of minutes before centrifugation at 16'100rcf for 1 minute at 21°C at step 10.

o The concentration of plasmid DNA obtained in each Eppendorf was measured at the NanoDropping machine.

The following concentrations (in ng/µL) were obtained:

o pBAD/AraC weak - 1. 186.4; 2. 211.4; 3. 170.6.

o pBAD/AraC strong - 1. 130.4; 2. 105.3; 3. 114.2.

o pLacI/Ara-1 promoter - 1. 280.7; 2. 272.2; 3. 299.2.

o (Double) terminator - 1. 76.8; 2. 60.9; 3. 78.0.

Preparation of the gel for Gel Electrophoresis involved:

o Adding 1 x 50mL TAE buffer to 0.5g agarose and microwaved until melted.

o Once cool, ethidium bromide (EtBr, 1µL) was added to the agarose gel and then mixed.

o The EtBr-containing agarose gel was then poured into an electrophoresis plate with a well comb inserted and left to set.

Each fragment was digested using the New England BioLabs BioBrick Assembly protocol for checking fragment sizes but using EcoRI-HF and PstI. The protocol was followed exactly, except:

o Each fragment was incubated with EcoRI-HF and PstI for 10 minutes.

o No heat-inactivation for 20 minutes at 80°C was used.

o Instead of ligation, 25µL each restriction digested sample was added to 5µL loading buffer.

o 25µL of each sample DNA was added to different wells, including DNA hyperladder (10µL).

o Gel electrophoresis was then run for approximately 40 minutes at 120V.

Gel Electrophoresis showed that pBAD/AraC weak, pBAD/AraC strong, pLacI/Ara-1 promoter and terminator were successfully cloned. Lane 1 = DNA hyperladder, Lane 2 = pLacI/Ara-1 promoter mini-prep 1 (expected 2 bands at: 2155bp for pSB1A3 and 103bp for the promoter), Lane 3 = pLacI/Ara-1 promoter mini-prep 2 (expected 2 bands at: 2155bp for pSB1A3 and 103bp for the promoter), Lane 4 = pLacI/Ara-1 promoter mini-prep 3 (expected 2 bands at: 2155bp for pSB1A3 and 103bp for the promoter), Lane 5 = pBAD/AraC weak promoter mini-prep 1 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 6 = pBAD/AraC weak promoter mini-prep 2 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 7 = pBAD/AraC weak promoter mini-prep 3 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 8 = pBAD/AraC strong promoter mini-prep 1 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 9 = pBAD/AraC strong promoter mini-prep 2 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 10 = pBAD/AraC strong promoter mini-prep 3 (expected 2 bands at: 2155bp for pSB1A3 and 130bp for the promoter), Lane 11 = DNA hyperladder, Lane 12 = (double) terminator mini-prep 1 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp), Lane 13 = (double) terminator mini-prep 2 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp), Lane 14 = (double) terminator mini-prep 3 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp), Lane 15 = (double) terminator mini-prep 1 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp), Lane 16 = (double) terminator mini-prep 2 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp), Lane 17 = (double) terminator mini-prep 3 (expected 1 band at 3189bp for pSB1AK3 as (double) terminator is too small to be seen at 95bp).