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Transformation and Golden Gate Assembly Training

Objective

Author(s): Pattarin Blanchard

Produce a recombinant strain of E. coli that appears as purple colonies. Do so by using 2024 Distribution Kit parts and Golden Gate Assembly to produce a construct. This multi-day experiment involves transformation, colony picking, plasmid purification, stock preparation, and assembly.

Construct Design

|GGAG| A A[Promoter: BBa_J23100] --> |TACT|B B[RBS: BBa_B0034_m1] --> |AATG|C C[CDS: tsPurple] --> |GCTT|D D[Terminator: BBa_B0015] --> |CGCT|Y -->

Backbone: pJUMP28-1A

If you are in the UTEX group, hopefully some of these parts ring a bell to you. We use the BBa_B0034 RBS and BBa_B0015 terminator in our constructs. BBa_J23100 is a strong promoter from the same family of Anderson promoters we use.

Why the BBa_B0034_m1 RBS? The m1 suffix indicates this variant of B0034 has a short spacer after the RBS sequence. According to the registry, generally RBSs require 6-7 base pairs for optimal translation efficiency, so the m1 variant is used over the minimal B0034 part, also found in the kit. Use this as a rule of thumb unless explicitly indicated in a part’s documentation.

Since these are from the distribution kit, they use the golden gate affixes prescribed in iGEM’s Type IIS standard, which allows them to be assembled in the desired promoter-RBS-CDS-terminator order. Parts in the distribution kit come in vectors so a minimal amount is shipped to us, meaning we are responsible for amplifying the parts prior to use.

TsPurple (short for tinselPurple) is a chromoprotein, meaning unlike fluorescent proteins like GFP, RFP, etc., it is visible without needing UV light. This makes chromoproteins See this page for more commonly-used chromoproteins and reporters in iGEM: .

Day 0: Simulating the assembly

pJUMP28-1A(sfGFP) can be downloaded from the Oligonucleotide/Gene Database.

For the rest, obtain plasmid sequences from the 2024 kit platemap: https://airtable.com/appgWgf6EPX5gpnNU/shrb0c8oYTgpZDRgH/tblNqHsHbNNQP2HCX

• Each of these should automatically be annotated, but for tsPurple you may want to manually label the translated feature yourself. Simulate Golden Gate Assembly using BsaI. The construct should look like this:

Day 1: Resuspension and transformation

Materials

• Competent E. coli cells, kept on ice

• Molecular water

• 8x Cm LB agar plates

• 2x Kan LB agar plates

• Sterile loop

• Floating foam tube rack

• 5x sterile microcentrifuge tubes

• LB medium, 1.5 mL aliquot

Equipment

• Water bath at 42C

• Bunsen burner

• Timer

• Ice bucket

The parts/plasmids can be retrieved from the following wells from the 2024 kit.

Name	ID	Plate	Well	Ab resistance	LABEL
Promoter	BBa_J23100	1	A5	Cm	1
RBS	BBa_B0034_m1 (BBa_J428038)	1	I19	Cm	2
tsPurple	BBa_K1033906	1	G1	Cm	3
terminator	BBa_B0015	1	C1	Cm	4
pJUMP28-1A	BBa_J428353	1	A10	Kan	0
pDest		2	G7	Amp	a

Preparation

The following take some time so they should be the first things you do, in addition to setting up your bench space.

1. Place agar plates in the BSC with the agar side on top and slightly off to dry and warm to room temperature
2. Place competent cells and empty microcentrifuge tubes on ice
3. Turn on the water bath and set the temperature to 42C.

Resuspending DNA

If this is the first time a part is being retrieved, follow these steps. Otherwise, simply thaw the distribution kit and take 1 uL of resuspension.

Reference protocol: Retrieving Parts from Distribution Kits

Transformation

Follow Heat Shock Transformation with a Water Bath.

For step 3, use 1 uL of resuspended DNA into its corresponding centrifuge tube with comp cells.

For step 6, use 200 uL LB medium (no AB needed) into each tube.

For step 7, incubate tubes in the 37°C room for 1 hour.

Note: The iGEM protocol for distribution kit parts says 2 hours incubation, but the time tends to vary between protocols. According to addgene, this step is to allow bacteria to produce Ab resistance proteins in advance.

For step 9, pipette 20 uL onto a corresponding plate and spread immediately with a sterile loop or glass beads. Label the plate with appropriate information, including the inocculation volume.

Note: The recommended amount varies by protocol and person. This is from the iGEM protocol for distribution kit parts.

Repeat step 9 on a second plate, this time with 200 uL. Having one plate with little and another with a lot maximizes the chance of getting a colony.

Incubate plates at 37C overnight.

*In a typical transformation experiment you would have four or more plates for positive and negative controls e.g. cell only, cell+backbone, no cell, cell+construct, but we will only do it for Day 3 because we already know these vectors in Day 1 should work.

Day 2: Colony picking

Materials

• 5x sterile glass or disposable culture tubes
• LB medium
• 1000x Cm and Kan stock
• Sterile 20/200uL pipette tips
• 25 mL serological pipette

Procedure

1. Thaw Cm and Kan stock on ice. The following steps should be done aseptically:

2. Add 4mL LB medium to each culture tube. Label as appropriate.

3. Once thawed, add 4 uL Ab stock to the corresponding tubes. From the table above, 1-4 should get Cm, 0 should get Kan.

4. For each plate, use a marker to circle the colony to be picked. > Note: Ampicillin (Amp) when used as a resistance marker tends to form satellite colonies that appear to have resistance but are just piggybacking off of a resistant colony, therefore when using Amp, pick the large colony in the middle instead of small ones around a larger one. Cm, Kan, and Spec do not exhibit this behavior so we do not need to worry about it.

5. For each plate, use a sterile pipette tip to gently touch the desired colony, and drop that pipette tip into the culture tube. Do not poke into the agar.

6. Bring the culture tubes over to the 37C room in a tube rack and place on a shaker for overnight culture. Continue the following day. # Day 3: Plasmid purification

Materials

• GeneJET miniprep kit
  • Resuspension Solution (Buffer P1)
  • Lysis Solution (Buffer P2)
  • Neutralization Solution (Buffer N3)
  • Wash Solution (Buffer PB)
  • RNase A
  • Elution Buffer (Buffer EB; 10 mM Tris-HCL, pH 8.5)
• 10x microcentrifuge tubes
• 5x collection tubes+spin columns (close the bag tightly afterward)

Miniprep & Quantification

1. Pre-label tubes. Each culture to be miniprepped gets two microcentrifuge tubes and a collection tube + spin column. Arrange them on a tube rack like so: ------microcentrifuge tubes (culture)------ ------empty------ ------collection tubes+spin columns------ ------empty------ ------microcentrifuge tubes (product)------

2. Proceed with Plasmid Mini-Preparation

3. Measure DNA concentration using NanoDrop Spectrophotometer.

Day 4: Golden Gate Assembly and Transformation

Golden Gate Assembly

Materials

• NEBridge ligase master mix
• DNA fragments (miniprepped DNA from the day prior) stored on ice
• Molecular-grade water
• BsaI stock (BsaI-HFv2), thaw on ice
• 1 PCR tube per replicate
• ice bucket (multiple if many people doing it)
• Thermocycler

Procedure

Reference protocol: Golden Gate Assembly with NEBridge Ligase Master Mix

1. Calculate how much each fragment is required to get 0.05 pmol, the sequence length of each fragment is tabulated below:

   Name	Label	Length (bp)
   Promoter	1	2424
   RBS	2	2411
   tsPurple	3	2736
   terminator	4	2520
   pJUMP28-1A	0	3359

2. Add the components according to the reference protocol.

3. For a 5-fragment assembly (like in this training session, including the backbone), follow the reference protocol for 3-6 fragment assembly.

4. Chill on ice once reaction is complete. Proceed to transformation.

Transformation

Materials

• LB medium
• Competent cell stock (start thawing in the middle of the GGA incubation cycle)
• Foam tube rack
• Water bath at 42C
• Glass beads
• Sterile microcentrifuge tube
• 4 Kan LB agar plates + each additional transformation replicate

Procedure

This time we will make proper controls for our transformation experiment, so there will be 4 plates per replicate:

cell+construct | cell+backbone

(positive control)
cell only
(negative control 1)	medium

(negative control 2)

1. Label and pre-chill centrifuge tube(s) on ice. Ensure the water bath is ready at 42C.
2. Make 1 mL aliquot of LB medium in a sterile microcentrifuge tube.
3. Pipette 50 uL of competent cells per tube and keep on ice.
4. Pipette 2 uL of GGA construct (or 1 uL pJUMP28-1A, part 0) into a centrifuge tube with comp cells. Close tubes and flick gently to mix, but do not vortex.
5. Incubate tubes on ice for 20 mins.
6. Using a floating tube rack, heat shock tubes in the 42C water bath for 45s, then rest tubes on ice for 3 mins.
7. Pipette 200 uL LB medium (no AB needed) into each tube.
8. Incubate tubes in the 37C room for 45 mins, preferably on a shaker or rotor.
9. Pipette 50 uL onto a plate and spread with 7-10 glass beads by moving the plate around horizontally (try not to let the beads touch the lid), then carefully deposit the beads in an ethanol-filled beaker (dirty beads beaker). Label the plate with appropriate information.
10. Stack the plates and tape, bring to 37C room to incubate plates overnight.
11. Close the microcentrifuge tubes well and dispose of them in the biohazard bin.

Day 5: done!

Upload pics of plates here so we have a running record.