Stanford/BIOE44:Module 1:Day4

M1: Day 4 - What happened?

Results

So things didn't go as well as we hoped. Only one group had colonies grow. But this is how research goes sometimes (a lot of the time?). We need to go back and think through what we did and at what steps things could have gone wrong.

Troubleshooting The Results

We will discuss and brainstorm on this topic in class.

Introduction to Arsenic Detoxification Genes in Bacteria

Arsenic is a well known environmental toxicant. For the purpose of this lab class you should be aware that arsenic is often found in one of two oxidation states: arsenite (+3) and arsenate (+5). Arsenic acts as a toxicant through two major routes. First, arsenate (AsO4), which is chemically similar to phosphate (PO4), can enter through a cell’s phosphate transporters embedded in the cell membrane. Once in the cell, arsenate can interfere with oxidative phosphorylation. Arsenite (AsO3) is also problematic. It inhibits crucial cellular enzymes, including the pyruvate dehydrogenase, which is central to glucose metabolism (Oremland 2003).

A diverse array of bacteria have evolved mechanisms for detoxification. A well characterized example is the ars operon. Fortunately for you, this genetic system is present in many lab strains of E.coli. While more complex ars operon’s exist, the simplest is composed of three main elements: arsB, arsC, and arsR.

They encode the following proteins

1. ArsB –an arsenite transporter.
2. ArsC – an enzyme catalyzing the reduction of arsenate to arsenite so that it can be excreted by arsenite transportersm like ArsC.
3. ArsR – a repressor protein that binds an operon upstream of the ars genes. It provides an example of a negative autoregulation in the absence of arsenic induction.

The Ars Operon.

For more information on diversity of bacteria and archaea that incorporate arsenic into their metabolism, see: Oremland, R. S. and J. F. Stolz (2003). "The ecology of arsenic." Science 300(5621): 939-944.

Building a New BioBrick Part

The standard biological part you are going to rebuild is Part: BBa_J33201 designed and added to the registry by the Edinburgh 2006 iGem team. You can view the part at [[1]]

The part includes the arsR gene and upstream regulation sequence. Recall that the part includes the nucleotide sequence for the arsR gene that encodes the ArsR protein. The ArsR protein is a repressor protein that binds to the operator sequence upstream of the arsR gene. The ArsR protein prevents the transcription of more copies of arsR mRNA under normal conditions. However, when arsenic is present, the metal binds to the ArsR protein, causing a conformational change that prevents ArsR from binding to the operator upstream of ars operon. As a consequence, DNA polymerase can transcribe both the arsR gene and the genes immediately downstream.

You could order this standard biologicak part from the registry, but you need to also know how to make it from scratch. Indeed the Edinburgh team did not have a full catalog at their disposal. How might you go about this? You should propose two distinct ways to recreate this part.One way can require tools not at your disposal. Another must be possible with the equipment you see in the lab.

1. Where might you find this genetic material?
2. How would you be able to get it out of a genome and into a plasmid?
3. Most importantly, how could you convert a gene to an easy to use biobrick part?

Brainstorm with your classmates and if you need help ask one of the TAs for some hints. Once you have proposed a way to build the part, come find a TA to get a sheet with some further guidelines.

• Please read:

In Class

Ligation

We will be following this protocol taken DNA ligation. You need to determine what your ligation reaction components and volumes should be. This ligation calculator will be helpful.

Ratios for 10μL Ligation Mix

Larger ligation mixes are also commonly used, but the ratio of insert to vector is the important thing.

• (1/10th total volume)μL 10X T4 ligase buffer
• 6:1 molar ratio of insert to vector
• Add (total volume - vector and insert volume)μl ddH₂O
• 0.5 μL T4 Ligase

Calculating Insert Amount

[math]\displaystyle{ {\rm Insert\ Mass\ in\ ng} = 6\times\left[\frac{{\rm Insert\ Length\ in\ bp}}{{\rm Vector\ Length\ in\ bp}}\right]\times{\rm Vector\ Mass\ in\ ng} }[/math]

The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.

• Assume your insert concentration (after gel extraction) is 200ng/uL.
• Assume your cut vector concentration (after gel extraction) is 200ng/uL.
• You cannot accurately pipette less than 0.5uL.

Protocol

1. Add appropriate amount of deionized H₂O to sterile PCR tube
2. Add ligation buffer to the tube.
   Vortex buffer before pipetting to ensure that it is well-mixed.
   Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
3. Add appropriate amount of insert to the tube.
4. Add appropriate amount of vector to the tube.
5. Add 0.5 μL ligase.
   Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.
6. Let your ligation sit at 22.5°C for 30 mins
7. Denature the ligase at 65°C for 10min. (This is important since intact ligase will interfere with transformation in the next step of the experiment).

Electrocompetent Cell Prep

The first two steps of the protocol have been done for you.

1. Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
2. Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C.
3. Measure the optical density (OD) of the culture by:
   1. Transferring 700uL of the culture to a cuvette for the spectrophotometer.
   2. Measure the absorbance at 600nm. Record this value. You want it to be between 0.50–0.60. This number is an indicator of what phase of growth the cultures are. We want them to be in log phase.
   3. If the number is too low, let them continue growing.
4. Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
5. Wash the cell pellet with 20 ml of ice-cold H2O then centrifuge again as above.
6. Resuspend the pellet in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 30 seconds.
7. Repeat step 6 twice more (minus the transferring to a new tube part).
8. Resuspend the cell pellet in 150uL and keep on ice.

Transformation via Electroporation

This procedure was adapted from a a full protocol by Knight

1. Aliquot 50uL of cells into pre-chilled 1.5mL tubes. (You'll have some leftover cells because when you resuspended it was really 150uL+the volume of the cells).
2. Add 0.5uL of DNA to each tube(from your ligation, ligation control and transformation control) and put them back on ice.
3. Get the following items together and then go over to the electroporator:
   • P1000 (set to 950uL)
   • P200 (set to 50uL)
   • In your ice bucket:
     • Tubes with cells+DNA
     • Three prelabelled 1.5mL tubes (corresponding to your tubes of cells+DNA)
     • Three electroporation cuvettes (from cold room)
4. At the electroporation station make sure there are pipette tips (1000s and 200s) and SOC.
5. Turn on electroporator, select preset protocols (#4), bacterial (#1), E.coli 1.8kV (#1).
6. Pipette the cell-DNA mixture between the two metal plates of the electroporation cuvette using your P200 pipetman. Try not to handle cuvette base (and metal parts) too much so that it stays cold.
7. Tap the cuvette on the counter so that cells are at the bottom and to remove any air bubbles.
8. Place in chamber of electroporator. Slide the cuvette in so that it sits snugly between electrodes. Close the lid.
9. Shock the cells by pressing the red button on electroporator. (Wait for the beep). A number will be displayed (the time constant) - you want this number to be between 4.3 and 5.0. Generally closer to 5.0 is better.
10. Remove cuvette from the chamber and immediately add 950uL of SOC . This step should be done as quickly as possible to prevent cells from dying off.
11. Transfer SOC-cell mixture to chilled prelabelled eppendorf tube.
12. Chill sample on ice for 2 mins to permit the cells to recover.
13. Transfer the tubes to 37°C incubator; shake to promote aeration (just tape them to the platform). Incubate for 30 minutes.

Plating

Now will be a great time to practice your sterile technique, so get out your Bunsen burner. You will need a LB-agar plate with Ampicillin and some sterile glass beads.

This is how you should plate.

1. Spin down your cells at 4000g for 2 minutes.
2. Resuspend your cells in 250uL SOC.
3. Label your plate. Pour approximately 15 glass beads on to your plate (Don't count just quickly transfer them to minimize contamination)
4. Pipet all of the cells onto the center of the plate and close the lid.
5. Place the plate on the bench and rapidly move the plate across the bench top at 90 degree angles. Don't swirl the glass beads as you won't spread your cells evenly (they'll end up all at the edges of the plate).
6. Once you believe the cell media has been spread across the plate, hold the plate vertically, so that the beads rest on the bottom edge. Slightly open the plate allowing the glass bead to fall into the bead waste beaker. Again, only open the plate slightly to minimize potential contamination.
7. Incubate plate overnight at 37°C. (Don't parafilm.)